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Corso di Dottorato in Chimica Industriale – XXIX ciclo
DESIGN, SYNTHESIS AND CHARACTERIZATION OF
MAGNETIC BIO-INORGANIC NANOSYSTEMS WITH
THERANOSTIC FEATURES
Tesi di Dottorato di:
Sara Mondini
Matricola R10574
Tutor: Prof.ssa Alessandra Puglisi
Co-tutor: Dott. Alessandro Ponti
Anno accademico 2015-2016
Ai miei genitori, Carmen e Giuliano
“Whatever you can do or dream you can, begin it.
Boldness has genius, power and magic in it!”
Johann Wolfgang von Goethe
TABLE OF CONTENTS
o Abstract pag 1
o Introduction pag 4
References pag 16
o Part I : Magnetic nanoparticles (NPs) coated with
zwitterions
01. Introduction pag 19
02. Preparation of iron oxide NPs coated with zwitterions
a. Synthesis of magnetic NPs pag 23
b. Catecholic ligands pag 26
c. Preparation of water-soluble NPs via ligand exchange
procedure pag 27
d. Characterization of NP coating pag 29
d.01. FTIR spectra
d.02. TGA curves
d.03. UV-Vis spectra
03. Study of NP colloidal stability pag 32
04. NP Cytotoxicity pag 37
05. Uptake experiments by HepG2 cells
e. Qualitative iron quantification pag 38
f. Quantitative iron quantification pag 39
06. Study of the intracellular NP fate pag 41
07. Conclusions pag 44
08. Experimental section
g. Materials pag 45
h. Procedures pag 45
h.01. Acronym used in the experimental section
h.02. Preparation of 9 nm iron oxide NPs
h.03. Preparation of 12 nm iron oxide NPs
h.04. Synthesis of zwitterionic dopamine sulfonate (ZDS)
h.04.1 Synthesis of dopamine sulfonate, DS.
h.04.2 Synthesis of zwitterionic dopamine sulfonate, ZDS.
h.05. Synthesis of fluorescent catecholic ligand ( FCL)
h.05.1 Synthesis of compound 2
h.05.2 Synthesis of compound 3
h.05.3 Synthesis of compound 4
h.05.4 Synthesis of compound 5
h.05.5 Synthesis of compound 6 ( FCL)
h.06. Ligand exchange procedures
h.06.1 Preparation of MEEA- or ZDS-coated NPs
h.06.2 Preparation of FCL-coated NPs
h.07. Evaluation of NP cytotoxicity
h.08. Evaluation of NP uptake by HepG2 cells
h.08.1 Qualitative determination of NP internalization
h.08.2 Quantitative iron evaluation
h.09. Intracellular iron localization
i. Characterization methods and instruments pag 55
References pag 56
o Part II : Magnetic NPs coated with functionalized
polyethylene glycol ( PEG)
01. Introduction pag 60
02. Preparation of iron oxide NPs coated with functionalized
polyethylene glycol
a. Synthesis of magnetic nanoparticles pag 67
a.01. Synthesis of magnetic NPs by hot-injection
a.02. Synthesis of magnetic NPs by co-precipitation
b. Synthesis of catecholic PEG ligands pag 70
b.01. Synthesis of catechol (a) and (b) compounds
b.02. Synthesis of catechol (c) compounds
c. Preparation of water-soluble PEG-ylated NPs pag 73
d. Characterization of PEG-ylated NPs pag 74
03. Conclusions pag 78
04. Experimental section
e. Materials pag 79
f. Procedures pag 79
f.01. Acronyms used in the experimental section
f.02. Synthesis of magnetic NPs by hot-injection
f.03. Synthesis of magnetic NPs by co-precipitation
f.04. Synthesis of the bi-functional linker (SS33)
f.04.1 Synthesis of compound 2
f.04.2 Synthesis of compound 3
f.04.3 Synthesis of compound 4
f.04.4 Synthesis of compound 5
f.04.5 Synthesis of compound 6
f.05. Synthesis of the catechol-PEG5000 co-surfactant (SS20_A)
f.05.1 Synthesis of compound 7
f.05.2 Synthesis of compound 8
f.06. Synthesis of the catechol (c) co-surfactant: synthesis of the
brominated PEG derivative 12
f.06.1 Synthesis of compound 10
f.06.2 Synthesis of compound 11
f.06.3 Synthesis of compound 12
f.06.4 Synthesis of the mono-catechol (c) co-surfactant (16)
f.06.5 Synthesis of the mono-catechol (c) co-surfactant:
tertbutyldimethylsilyl (TBDMS) ether protecting group
f.06.6 Synthesis of compound 18
f.06.7 Synthesis of compound 19
f.06.8 Synthesis of compound 13_TBDMS
f.06.9 Synthesis of compound 14_TBDMS
f.06.10 Synthesis of the mono-catechol (c) co-surfactant:
investigation of reactivity of 14_TBDMS
f.06.11 Synthesis of compound 21
f.06.12 Synthesis of the mono-catechol (c) co-surfactant:
acetonide protecting group
f.06.13 Synthesis of compound 24
f.06.14 Synthesis of compound 25
f.06.15 Synthesis of compound 26
f.06.16 Synthesis of compound 13_Acetonide
f.06.17 Synthesis of compound 14_Acetonide
f.06.18 Synthesis of the mono-catechol (c) co-surfactant: benzyl
ether protecting group
f.07. Preparation of PEG-ylated NPs
f.07.1 Preparation of PEG-ylated NPs from those obtained by hot-
injection
f.07.2 Preparation of PEG-ylated NPs from those obtained by co-
precipitation
g. Characterization of PEG-ylated pag 93
g.01. DLS of PEG-ylated NPs
g.02. CHN analysis data
g.03. Fluorescent assay to quantify maleimide groups
h. Characterization methods and instruments pag 97
References pag 98
o Acknowledgements pag101
1
o Abstract
The aim of my PhD project is to obtain magnetic bio-inorganic nanosystems having both diagnostic
and therapeutic functions. We have focused our attention on magnetic nanoparticles (NPs) since
their properties allow to use them in diagnosis, as good MRI contrast agents and in therapy, as
colloidal mediators for hyperthermia or carriers for drug delivery. Furthermore, their diagnostic
properties could also allow one to monitor the response to therapy.
However, to achieve active targeting of magnetic nanocrystals in medical applications, we need to
study in detail their interactions with biological systems. Indeed, when nanoparticles are injected
into the bloodstream (we consider the parenteral administration because all cells receive supplies
thanks to the blood circulation), circulating plasma proteins, opsonins, adsorbe on the NP surface
forming a layer known as protein “corona”. Such protein layer is recognized by the cells of the
mononuclear phagocyte system (MPS), which remove the NPs from the blood by phagocytosis.
Usually, the MPS concentrates the NPs in organs such as the liver and the spleen and prevents the
NPs to reach their target. For this reason, one must rationally design nanosystems minimizing or
delaying their internalization by MPS cells in order to increase their plasma half-life and
consequently the ability to reach the desired organ/tissue by active targeting. Such NPs are known
as “stealth NPs”. The NP surface properties appear as more important than those of the core to
escape phagocytosis, because the coating is in direct contact with blood and organs. In particular,
during my PhD I have explored some examples of the two types of NP coating that, until now, have
been mainly studied to prevent protein adsorption: the “conventional” polyethylene glycol (PEG)
ligands and recently introduced small zwitterionic surfactants.
Therefore, in the first part of my PhD thesis, we reported on magnetic iron oxide NPs coated with
zwitterionic dopamine sulfonate (ZDS), containing a sulfobetaine group that we have chosen among
the zwitterionic groups used in nanomedicine. We have compared these NPs to similar NPs coated
with PEG-like 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (MEEA) to investigate how surface
chemistry affects the NP in vitro behavior. ZDS-coated NPs showed a very dense coating, that
ensures high colloidal stability to the nanosystems in several aqueous media and negligible
interaction with proteins. Treatment of HepG2 cells with increasing concentrations (from 2.5 up to
100 µg Fe/ml) of ZDS-NPs had no effect on cell viability and resulted in a low, dose-dependent NP
uptake, lower than most reported data for the uptake of iron oxide NPs by HepG2 cells. Conversely,
MEEA-coated NPs showed lower ligand density and formed micrometer-sized aggregates in
deionized water. Such NPs had a cytotoxic effect for dose ≥ 50 µg Fe/ml, and were about 20-fold
2
more efficiently internalized than ZDS-coated NPs. We could conclude that the high-density ZDS
layer prevented the biocorona formation and both aggregation and sedimentation of iron oxide NPs.
Moreover, the very low internalization of ZDS-coated iron NPs inside HepG2 cells could be useful
to achieve an active targeting upon specific functionalization. We have also explored the
intracellular fate of ZDS-coated NPs, demonstrating that in HepG2 cells they are sent to the
lysosomial pathway.
The second part of this Thesis concerned NPs coated with PEGylated ligands. Considering the
well-known ability of the catechol (1,2-dihydroxyphenyl) group to bind iron ions with high affinity
and give stable NP water dispersion, we chose this grafting group for our NP ligands based on a
PEG 5000 polymer chain. We would also functionalize magnetic NPs with a large biomolecule, the
Fab fragment of Trastuzumab (Herceptin®), that is a monoclonal antibody selectively binding the
human epidermal growth factor receptor 2 (HER2), hoping in this way to endow the nanoparticles
with active targeting functionality because the antibody fragment strongly binds to its antigen.
Therefore, we have designed different catechol molecules: (i) a bi-functional PEG ligand displaying
a maleimide end-group able to react with the free thiol group of the Fab biomolecule; (ii) mono and
bi-dentate OMe-terminated PEG ligands in order to employ them as co-surfactants to dilute the
maleimide ligand, making the active molecule less sterically hindered while preserving water
solubility and colloidal stability of the system. With poly-dentate structures, we wished to have an
improvement in NP water stability by increasing the number of grafting groups per molecule and so
decreasing the probability that a ligand is irreversibly lost from the NP surface. Such structures are
based on ethylenediamine as a linker between the hydrophilic PEG chain and the catechol grafting
groups. The PEG chain is linked to one of amine groups of the scaffold and the other amine group is
linked to one or two 3,4-dihydroxybenzyl groups.
We chose to link the PEG chain to the central ethylenediamine scaffold as the last reaction step by
coupling it to the mono- or di-catechol-diamine moiety since the isolation and characterization of
the intermediate products are simplified. We have tested some protecting groups for catechol
moieties such as tert-butyldimethylsilyl (TBDMS) ether or acetonide, but we have found that both
these protecting groups might be – at least partially – deprotected in our reaction conditions. So we
finally tried the benzyl ether protecting group, more stable in our reaction conditions since it is
removed by hydrogenation. In addition to these synthetic studies, which are still in progress, I
prepared and characterized two batches of iron oxide NPs coated with a mixture of the catechol-
PEG-maleimide and catechol-PEG-OMe ligands. I synthesized one batch of NPs by solvothermal
method, while the other batch was produced by alkaline co-precipitation from a stoichiometric
3
aqueous solution of FeCl3 and FeSO4. Solvothermal NPs are highly monodisperse and crystalline,
whereas co-precipitated NPs can be produced in larger amount and in “green conditions”,
minimizing the use of toxic reagents, and are free from coating ligands. In both cases, the final yield
in water soluble NPs was around 50%. Indeed, the two batches of NPs were coated by different
amount of PEG ligands; the coating was more efficient for NPs produced by co-precipitation as
evaluated by CHN analysis. Finally, having the aim to functionalize the nanosystems with the Fab
fragment of Trastuzumab, I have quantified the reactivity of maleimide groups of the bi-functional
linker present in the NP coating. In particular, I carried out such quantification with a commercially
available fluorescent kit able to measure protein maleimide groups. By comparison with a shorter
maleimido PEG (molecular weight ≈ 2000 Da), I concluded that this moiety was less reactive in the
longer PEG5000 chain. However, a fraction of maleimide group exposed at the NP surface was
actually reactive and sufficient to functionalize the NCs with the desired amount of anti-body
fragment.
Therefore, both ZDS and PEGylated NPs could be considered promising in the development of
theranostic nanosystems.
4
o Introduction
A possible definition of “nanomedicine” is “the application of nanotechnology (the engineering of
materials with sizes in the nanometer range) in a healthcare environment, with the aim to prevent
and treat diseases in the human body”.
In general, a favorable prognosis is closely correlated to the detection and treatment of illness at an
early stage of development. Therefore, huge efforts have been made by biomedical researchers in
order to improve the sensitivity and accuracy of imaging techniques and the efficacy of medical
cares. Recently, nanotechnologists have designed a variety of theranostic nanosystems, where the
word “theranostic” was coined by Funkhouser1 in 2002 to define a material that combines
therapeutic and diagnostic modalities within a single platform.
These nanosystems can be composed of either organic or inorganic materials.
Organic platforms comprise polymer-drug conjugates2 such as poly(lactic-co-glycolic acid) (PLGA)
nanoparticles3, polymeric micelles
4, liposomes
5, high-density lipoproteins
6 and dendrimers and are
mainly used for drug delivery and gene therapy. In these cases, the drug or the gene are entrapped,
fixed, adsorbed, or enclosed into or onto the nano-matrices. Such systems increase drug efficacy
and are less toxic with respect to traditional drugs, so several of them are being tested in clinical
trials thanks to promising pre-clinical results such as those obtained using INGN-401, a liposomal
formulation to deliver a cancer-suppressing gene against metastatic lung cancer. In some cases,
organic nanosystems have already reached the market as Doxil®, a liposomal system to deliver
doxorubicin for the treatment of patients with ovarian cancer, AIDS-related Kaposi's sarcoma, and
multiple myeloma.
If we compare organic and inorganic nanoparticles (NPs), the latter are characterized by peculiar
physical properties that often are different from those of the bulk materials, thanks to (quantum)
size effects. For example, quantum dots (QDs)7-8
, dye-doped silica NPs9, and upconverting NPs
10
are very useful NP fluorescent markers thanks to their advantageous features arising from quantum
confinement effects at the nanometric scale. The ferrimagnetic iron oxides, i.e., magnetite or
maghemite, having diameter < 30 nm are in the so-called superparamagnetic regime, where their
permanent magnetization changes orientation very rapidly, thus featuring appealing relaxation
properties for use a MRI contrast agents..
In particular, we decided to focus our attention on magnetic NPs considering that, excluding
humans whose body contains magnetizable material (such as medical devices with batteries or
computer chips, vascular or intracranial metals), magnetic fields are not contraindicated for
patients11
and since, due to their special features, magnetic NPs appear as ideal platform materials
for theranostics12-13-14
.
5
First, magnetic nanocrystals (NCs) can be synthesized with controlled size that normally is in the
range from few nanometers up to tens of nanometers. They thus are smaller than cells (10-100 m),
larger than most organic molecules, and similar in size to viruses (20-450 nm) and biological
macromolecules such as proteins (5-50 nm), and genes (2 nm wide and 10-100 nm long), as
depicted in Fig 115
. For this reason, magnetic NPs can interact with biological objects, labeling them
and potentially taking part in metabolic processes.
Fig 1. Comparison between the dimensions of magnetic nanoparticles and biological entities.
Image from Ref 15.
Second, both the magnetic NP core and shell can be made of non-toxic material. For example, iron
oxide NPs are usually employed in biomedical field for their biological compatibility. Moreover, in
addition to biocompatibility, other functions can be inserted in their tailor-made surface coating,
such as the ability to prevent the formation of the protein “corona”, selectively bind to a specific
biological entity or deliver compounds and drugs to defined sites.
Third, the permanent magnetic moment of magnetic NPs provides further functionalities. The above
mentioned superparamagnetism makes magnetic NPs excellent MRI contrast agents because they
strongly contribute to the relaxation of the hydrogen spins in water molecules. Furthemore the NP
magnetic moment can be manipulated using an externally applied magnetic field. For instance, a
magnetic field gradient can be used to separate NPs or localize them in particular regions of the
body. Another example is based on the interaction of the magnetic NPs with an alternating magnetic
6
field (radiofrequency) which heats the NPs and can then be exploited in the field of hyperthermic
therapy.
Magnetic NPs for separation devices
A magnetic field gradient can exert a force at a distance adequate to move magnetic NPs tagged, at
their external coating, with molecules or biological entities which in this way could be separated
from their native medium. Such magnetic separation can be obtained by passing the fluid mixture
through a region where a magnetic field gradient immobilizes the magnetic NPs. In this simple
device ( Fig 212
) a magnet fixed to the container wall of a solution can collect the tagged particles,
while the unwanted supernatant solution is removed.
Fig 2. Standard method of magnetic separation of magnetically tagged (•) and unwanted (◦)
biomaterials. Image from Ref 12.
The main limitation of this simple technique is the slow accumulation rates of the tagged NPs.
Therefore other more complex magnetic separation devices are developed.
Magnetic NPs as colloidal mediators for hypertermia
In addition, an alternating magnetic field of sufficient strength and suitable frequency can permeate
human target tissues and transfer energy to magnetic NPs, in order to heat them up. The
environment close to the NPs is also heated by thermal conduction. This principle allows one to use
NCs as agents for magnetic fluid hyperthermia, providing a promising therapeutic solution to the
homogeneous treatment of deep or scattered cancers. In fact, hyperthermia is used to destroy
malignant cells11
in combination with other cancer therapy such as chemo- and/or radio-therapy
since its local application can reduce side effects of treatment, such as systemic effects. The
temperature is maintained in the range of 42-45 ˚C for 30 min or more, because it is known that
tumor cells are more sensitive to temperature in this range than normal tissue cells. Magnetic NPs
are appealing agents since they allow clinicians to heat only the target tissue (Fig 3) without
damage of surrounding healthy cells.
7
Fig 3. Animal trial data on hyperthermia treatments in rabbits,showing preferential heating of a
tumour using intra-vascularly infused ferromagnetic microspheres; tumour edge, (♦) tumour
centre, normal liver 1–2 cm from tumour, (×) alternative lobe, and core body
temperature. Image from Ref 12.
There are different types of hyperthermia depending on the size of the heated body part. In local
hyperthermia heat is applied to a small area while in regional hyperthermia a large tissue portion
(such as a body cavity or organ) is heated; whole-body hyperthermia is used to treat metastatic
cancer that has spread throughout the body. The efficacy of hyperthermia therapy is connected to
the temperature achieved during the treatment, as well as to the duration of treatment and cell and
tissue features. To ensure that the desired temperature is reached without excess heating, the
temperature of the tumor and surrounding tissue is monitored during the treatment by thermometers
inserted inside small needles or tubes. Many clinical trials are being managed to evaluate the
efficacy of hyperthermia. Magnetic hyperthermia is a promising type of hyperthermia which
exploits the ability of small magnetic particles to strongly absorb electromagnetic radiation in the
radiofrequency-microwave range and to transform it into heat. Magnetic hyperthermia is effective
since radiofrequency-microwave easily penetrate tissue, well localizable (magnetic particles can be
located in the region to be treated) and free from radiation-related side effects. Magnetic
hyperthermia is currently in clinical use. MagForce is a company founded in 1997 that has launched
in 2011 the first and only nanotechnology-based therapy (NanoTherm™) currently having the
European regulatory approval (CE conformity marking) for the treatment of brain tumors. As
8
clearly described on their website, NanoTherm™ therapy employs magnetic NPs as elements able
to absorb electromagnetic radiation, . in particular NPs with size of 15 nm, coated by aminosilane
which endows them with water-solubility. These NPs, once introduced by injection into a solid
tumor, remain in the cancerous tissue for some time and act as transducers, able to convert the
energy coming from the alternating magnetic field applicator, the NanoActivator®, into heat.
NanoActivator® devices (Fig 4) are installed in hospitals in Berlin, Münster, Kiel, Cologne and
Frankfurt. can be used for tumors in all regions of the body. Indeed, NanoTherm® Therapy was
already clinically tested on approximately 90 patients with brain tumors and about 80 patients with
other tumors such as pancreatic, prostate or esophageal cancer.
Fig. 4. NanoActivator® (from http://www.magforce.de/en/produkte/nanothermr.html).
Magnetic NPs for drug delivery
In cancer therapy, magnetic NPs can be also coupled with targeting agents and therapeutic drugs,
with the aim to enhance treatment specificity and to reduce general side effects of chemotherapies.
Therefore, they are used as magnetic carriers to transport and release the active molecule at the
desired tissue. For example, iron oxide NPs covalently conjugated with methotrexate (MTX), a
chemotherapeutic drug able to cause apoptosis of malignant cells, can be addressed to tumor cells
which over-expressed folate receptors such as human breast cancer cells (MCF-7), human cervical
cancer cells (HeLa), 9L glioma cells16-17
. Cancer cells internalized a higher level of functionalized
NPs than healthy cells and the MTX release was obtained under low pH conditions through the
action of intracellular enzymes within the lysosomal compartment (Fig 5).
9
Fig 5. Schematic representation of the intracellular uptake of MTX modified NPs into breast cancer
cells. After a receptor-mediated endocytosis, magnetic NPs are transported to early endosomes
which then fuse with lysosomes containing proteases, which cleave the peptide bond between the
NP and the drug, allowing its delivery into the target cell. Image from Ref 16.
Some interesting results have also been obtained using iron oxide NPs conjugated to the epidermal
growth factor receptor (EGFR) inhibitor cetuximab against malignant gliomas, the most common
and lethal primary brain tumor that frequently over-expresses EGFR receptor18
. In this case, the
treatment with -loaded NPs has an antitumor effect greater than with cetuximab alone.
Porous hollow NPs (PHNPs) of Fe3O4, produced by controlled oxidation of Fe NPs at 250 °C
followed by acid etching, were filled with cisplatin by diffusion. They can release cisplatin at low
pH condition. After coupling with Herceptin to the surface, the cisplatin-loaded hollow NPs
provided efficient delivery of cis-platin to breast cancer SK-BR-3 cells with IC50 reaching
micromolar concentration much lower than that needed for free cisplatin19
.
Magnetic NPs as contrast agents
Finally, magnetic NPs are employed as contrast agents for magnetic resonance imaging (MRI), a
diagnostic technique usually used for three-dimensional non invasive scans of the human body.
According to different relaxation pathways, MRI images can be classified as T1- (longitudinal
relaxation time)- or T2- (transverse relaxation time)-weighted images. MRI contrast agents can
10
shorten both T1 and T2, helping to improve contrast in MRI images and to ease image interpretation
by clinicians.
Magnetic NPs are T2 contrast agents because they shorten T2 when, under the application of a
magnetic field, their magnetic moment quickens the magnetic relaxation processes of the water
protons close to the NPs in the body tissue. Such changes result in darkening of the corresponding
area in T2-weighted MR images. The NP behavior in vivo is a significant challenge associated with
their application as contrast agents. As better explained in the next section of the Introduction, all of
these magnetic NPs are almost non-specifically taken up by the mononuclear phagocyte system
(MPS) since they are recognized as not-self by the body, also in consideration of their size. NPs
having diameters of ca 30 nm or more are rapidly collected by liver, spleen, and lymph nodes
decreasing their effectiveness as MRI contrast agent, while particles with sizes of ca 10 nm or less
are not so easily recognized by the MPS. The next generation of NP-based contrast agents should
incorporate novel nanocrystal cores, coating materials, and functional ligands to improve their
contrast effectiveness and specific delivery/targeting. In this way, they would also allow one to
perform real-time monitoring of the pharmaceutical treatment. For example Herceptin®, that is an
antibody specifically binding to the over-expressed HER2/neu marker on the surface of breast and
ovarian tumors, was coupled to a series of metal-doped NPs having spinel structure MFe2O4 where
M is a divalent cation such as Mn, Fe, Co or Ni20
. As shown in Fig 6, MnFe2O4 NPs (MnMEIO)
exhibited higher MRI sensitivity than Fe3O4 NPs (CLIO), consistent with the magnetization results.
11
Fig 6. In vivo MR detection of cancer using magnetic NP–Herceptin conjugates. Color maps of T2-
weighted MR images of a mouse implanted with the cancer cell line NIH3T6.7, at different time
points after injection of 50 mg of MnMEIO-Herceptin conjugates or CLIO-Herceptin conjugates. In
a–c, gradual color changes at the tumor site, from red to blue, indicate progressive targeting by
MnMEIO-Herceptin conjugates. In contrast, almost no change was seen in the mouse treated with
CLIO-Herceptin conjugate (d–f). Image from Ref 20.
Magnetic NP plasma half-life and biodistribution
The use of magnetic NPs in nanomedicine requires a detailed knowledge of their interactions with
the biological system.
In particular, one of the key aspects that influences the biodistribution and the biocompatibility of
theranostic nanosystems throughout the body is protein binding21
. Biologically active materials or
NPs are often introduced into an organism via parenteral administration because all cells receive
supplies thanks to the blood circulation. After the introduction into the physiological environment,
the surface of the materials or NPs are bound by proteins, which form an adsorbed layer known as
the protein “corona”. The NP-protein complex is the dynamic outcome of multiple interplaying
12
factors. Several plasma proteins (immunoglobulins, apolipoproteins, clotting factors and
complement proteins) have been identified bound to various NPs. In particular, it is known that the
presence of certain plasma proteins, called opsonins, on the NP surface creates a “molecular
signature” which is recognized by immune cells of the mononuclear phagocyte system (MPS),
previously known as reticulo-endothelial system (RES). MPS comprises monocytes, which
circulate in the blood, dendritic cells and macrophages present in tissues like the liver (Kupffer
cells), spleen, lungs, and bone marrow22
. Therefore, the opsonization process enhances the NP
uptake from the bloodstream by MPS cells through endocytosis/phagocytosis and their
concentration in organs with high phagocytic activity (Fig 7). Indeed the NP accumulation can
induce tissue inflammation and release of toxic byproducts if the NPs decomposed.
Fig 7. Schematic representation of biomolecular corona formation and its influence on active
targeting. Under serum-free in vitro conditions, active targeting of functionalized NPs is achieved
by interaction and recognition of cell membrane-located receptors with targeting moieties grafted
onto the NP surface. In a biological milieu, a biomolecular corona is formed by non-specific
adsorption of proteins and lipids to the NP surface. This process impairs active targeting and leads
to NP accumulation in the tissues of the MPS. Image from Ref 22.
13
After i.v. injection, colloidal drug carriers are removed from the bloodstream within minutes and
their typical final biodistribution is of 80–90% in the liver, 5–8% in the spleen and 1–2% in the
bone marrow23
. In fact Kupffer cells together with macrophages in the spleen rapidly uptake NPs
with hydrodynamic diameters larger than approximately 100 nm24
. Instead NPs with diameters of ≤
5 nm are efficiently filtered by kidneys through their glomerulus pores (that have diameters around
10 nm) and are thus rapidly cleared from the blood25-26
(Fig 7).
So, it is clear that the protein corona affects the further NP biological destiny inside the body,
changing its rate and route of clearance from the bloodstream.
Other features that influence the biodistribution within the body, in particular of NPs with
dimensions ranging from 5 to 100 nm, are size27
, shape28
, solubility, surface charge (as measured by
zeta potential), coating functionalization, and route of administration. For NPs of the same
composition, lower amounts of proteins are adsorbed onto smaller particles in comparison to larger
particles, which are easily captured by MPS.
NPs having a sufficiently long half-life can accumulate inside the tissues in different ways
depending on the morphology of the epithelium near their blood vessels. Such aspect could be
effective in the cancer imaging and treatment since most solid tumors exhibit a “leaky” vasculature
with structured fenestrations, due to the high metabolism rate of their cells. This feature, along with
poor lymphatic drainage, endorses the passive accumulation and targeting of NPs with size between
30 and 200 nm within the tumor and it is called “enhanced permeability and retention (EPR)
effect”. In addition to the passive targeting, most NPs are captured by cells via endocytosis that
could be receptor mediated (the mechanism most efficient for NPs of 50 nm diameter), caveolae
mediated or adsorptive (Fig. 8)22
.
14
Fig 8. Graphical representation of the major size-dependent biological barriers that influence the
biodistribution of blood-circulating NPs. (A): Phagocytosis of NPs with hydrodynamic diameters
>100 nm by Kupffer cells in the liver. (B): Glomerular filtration and urine excretion of NPs smaller
than 5.5 nm. (C): “Enhanced permeability and retention (EPR) effect”of NPs with hydrodynamic
diameters between 30–200 nm in a large and well-vascularized tumor. (D): NP internalization
mechanism by cells. Key: (i) endothelial cell; (ii) Kupffer cell; (iii) hepatocyte; (iv) glomerular
basement membrane; (v) tumor cell. Image from Ref 22.
Therefore, researchers have to rationally design nanosystems in order to use them in the body, if
they want to achieve active targeting. In particular, they have to minimize or delay the NP uptake
by MPS cells in order to increase the NP plasma half-life as long as possible and consequently the
probability of reaching the desired tissue. Such NPs could be named as “stealth NPs” .
With this aim, surface properties are more important than those of the core, because the coating is in
direct contact with the blood and organs.
Anti-fouling and biocompatible coatings
Protein-repellent surfaces are characterized by electrical neutrality, hydrophylicity and the ability
to play the role of hydrogen-bond acceptor, but not of hydrogen bond donor.
15
Until now, two types of NP surface modification have been mainly studied to prevent protein
adsorption, the “traditional” polyethylene glycol (PEG) coating and zwitterionic molecules. Both of
them are highly effective.
Protein adsorption can occur if the total free energy of the process is negative (ΔGads = ΔHads -
TΔSads < 0)29
. Since the enthalpic contribution is positive (the absorption of lysozyme30
or bovine
albumin, BSA, onto silica31
were reported to be endothermic processes), the driving force of the
process has to be entropic. For both PEG and zwitterion coatings the driving force has been
attributed to the release of counterions, according to the mechanism reported for the complexation
of proteins with charged polymers (see Fig 9). But for a neutral surface, such as a zwitterion or PEG
group, the surface charge is internally balanced (zwitterion) or neutral (PEG) and formation of an
ion pair with the adsorbate is improbable; therefore no ion is available for release from the surface
and consequently no protein adsorption occurs.
Fig 9. Cartoon for the ion-coupled adsorption mechanism of a protein with a net positive charge
onto a negatively charged hydrophilic substrate. (Top) Adsorption of protein is facilitated by the
release of counterions and formation of ion pairs between the sorbent and the adsorbate. (Bottom)
Neutral surface (zwitterion or PEG) has no surface ions associated with it. The binding of protein
to the surface will not result in a net increase in entropy due to counterion release, and thus,
adsorption is not preferred. Note that some of the charge is still associated with the original surface
but is inaccessible due to a steric barrier. Image from Ref 29.
16
Obviously, PEG and zwitterionic coatings show several differences mainly due to the largely
different molecular size. For example, the zwitterions, due to their small size, produce a thin coating
(a few nm thick) that does not much increase the size with respect to the uncoated NP, an important
factor related to the NP stealthiness and feasibility of renal excretion. Long-chain PEG, with
molecular weight > 1000 as normally used, forms a thick coating on the nanocore significantly
increasing the NP size. This is particularly important when the hydrodynamic size is considered,
since the PEG chain flexibility can lead to an increase of the NP hydrodynamic diameter by several
tens of nm32
. As we will describe in more detail in the following of this PhD thesis, we have
analyzed both classes of PEG and zwitterion surfactants, starting from a literature overview.
References
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H. G., Suh, J.-S., Cheon, J. Artificially engineered magnetic nanoparticles for ultra-sensitive
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18 Kaluzova, M., Bouras, A., Machaidze R., Hadjipanayis, C. G, Targeted therapy of glioblastoma
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H., Graham, B., Zwitterionic-Coated “Stealth” Nanoparticles for Biomedical Applications: Recent
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Renal clearance of quantum dots, Nat. Biotechnol., 2007, 25, 1165-1170.
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29 Estephan, Z.G., Schlenoff, P.S., Schlenoff, J. B., Zwitterion as an alternative to PEGylation,
Langmuir, 2011, 27, 6794-6800.
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32 Mondini, S.; Drago, C.; Ferretti, A. M.; Puglisi, A.; Ponti, A. Colloidal stability of iron oxide
nanocrystals coated with a PEG-based tetra-catechol surfactant, Nanotechnology, 2013, 24, 105702.
19
Part I: Magnetic NPs coated with zwitterions
01. Introduction
Recently, zwitterionic ligands have been used as an alternative to PEGylated coatings, aiming at
obtaining biocompatible nonfouling materials, able to prevent nonspecific protein adsorption to the
NPs1-2
. Some of the zwitterion functional groups more commonly employed in nanomedicine are
aminoacid (e. g. cysteine), carboxybetaine, phosphorylcholine, and sulfobetaine (Fig 1).
Fig 1. Zwitterionic functional groups. Image from Ref 1.
Cysteine is a zwitterionic amino acid and its thiol terminal group was used to stabilize NPs made
from semiconductors, such as CdS3, ZnS
4, and of noble metals Au
5 or Ag
6. Cysteine is not able to
prevent noble metal NP aggregation when the electrolyte concentration increases while it is
efficient with semiconductor NPs7 maybe thanks to a greater stability of the thiol-semiconductor
over the thiol-gold bond. In this work, Bawendi and co-workers showed that quantum dots coated
by cysteine and with a diameter less than 5.5 nm were collected in the bladder of rats since mainly
excreted by kidneys. However, the cysteine inclination to spontaneous oxidation led to NP
aggregation a few hours after preparation. A novel strongly-binding zwitterionic disulfide was used
to prepare small (average diameter around 4 nm) water-soluble Au NPs by replacement of the
pristine citrate ligand8. These NPs are stable in saline media with 3 M salt concentration and also
when charged polyelectrolytes or biopolymers were added to the aqueous NP solution, witnessing
the absence of nonspecific interactions. Afterwards, a siloxane sulfobetaine ligand was employed by
Schlenoff and co-workers to coat and stabilize silica9-10
and iron oxide11
NPs by siloxane
condensation chemistry, avoiding an appreciable increase of their hydrodynamic diameter. The
20
zwitterionic silica particles did not aggregate when exposed to aqueous 3 M NaCl and 50% FBS up
to 24 h. The authors varied the zwitterionic siloxane concentration in order to produce iron oxide
NPs small enough for renal clearance capability. They also demonstrated the NP stability in
aqueous solution in the pH = 6–9 range, while the tendency of NPs to aggregate at lower pH was
attributed to the presence of some pH-sensitive Fe-OH functionalities on the NP surface due to
incomplete capping.
In the literature it was also proved that sulfobetaine polymers, in contrast to classic polyelectrolytes
with a single type of ionized group, had a very small disturbing effect on the water hydrogen-
bonded structure12
. So their application was strongly advised in biomedical fields, e. g., iron oxide
NPs coated with polymeric sulfobetaines have already been described13-14
.
Indeed, larger-size NPs coated with zwitterions have shown an extended circulatory lifetime which
makes them ideal candidates as blood pool imaging agents. However, NPs with hydrodynamic
diameter less than 5 nm are sufficiently small to be filtrated through kidneys and eliminated via the
urine, a desirable feature for example if the NP is radioactively labeled15
. In fact, a safe and
successful NP application relies on a balance between a prolonged blood circulation (required to get
the NPs to the desired target) and an effective clearing mechanism (enabling minimization of side
effects due to a prolonged NP presence in the body, such as a deposition in non-targeted tissues and
organs)10
. To this purpose, an increase in NP size after the binding to the desired target, for example
20 h following the NP administration, can be useful for their elimination10
.
Considering the advantage which can arise from the small sizes of these zwitterionic ligands, we
focus our attention on small zwitterionic molecules effective in coating nanocrystals while
increasing their diameter only by a few nanometers.
The production, trough a nanoemulsion method, of a ultrathin coating layer of zwitterionic
molecules adsorbed via hydrophobic and van der Waals interactions at the surface of oleic acid-
NPs16
has been reported (Fig 2). In this way, Lee et al obtained water-soluble NPs having high in
vivo stability, as demonstrated by incubation in 1 M NaCl and 10% FBS solution. Moreover, the
ultrathin coating layer led to a measured T2 relaxivity much higher than that of commercial T2
contrast agents.
21
Fig 2. (a) Schematic of ZSPION synthesis and chemical structure of zwitterion surfactant. (b)
Photograph of dispersion behavior of oleic acid stabilized SPIONs before and after the zwitterion
surfactant coating in hexane/distilled water. (c) Hydrodynamic size distribution along with TEM
image of ZSPIONs. The scale bar in the TEM images denotes 20 nm. Image from Ref 16 .
Furthermore, small sulfobetaines have been reported to graft to NP surfaces. Bawendi and co-
workers synthesized and employed a novel dopamine sulfonate ligand to coat iron oxide NPs17
. As
the authors explained, the structure of the compound (see Fig 3) was designed considering that: (i)
the catechol unit strongly binds the iron centers of NPs18
, (ii) the sulfonate moiety ensures high
water solubility and (iii) in association with a quaternary amine group leads to a zwitterionic feature
which provides pH stability to the nanosystem and reduces the nonspecific protein adsorption.
Water-soluble ZDS-NPs, having a hydrodynamic diameter of 10 nm, were well dispersible in
phosphate-buffered saline (PBS) and stable in the pH range between 6 and 8.5 and with respect to
time and salinity. As expected from their zwitterionic nature, a reduced amount of serum protein
bound these NPs in comparison to negatively-charged iron oxide NPs. Finally the authors
22
functionalized ZDS-NPs with streptavidin and fluorescent dyes for biotin-specific targeting and
imaging (Fig 3).
Fig 3. Functionalization of iron oxide NPs coated with dopamine zwitterion (ZDS) ligand whose
chemical structure is reported in the yellow square. Image from Ref 18.
ZDS-coated NPs have a saturation magnetization (Ms) of 74 emu g-1
preserving the characteristics
of pristine iron oxide NPs from which they were obtained by a ligand exchange process19
. In the
same report, the authors also tested in vitro NP uptake by HeLa cells and they observed a low
aspecific cell internalization after 24 h incubation. In addition, they carried out an in vivo stability
experiment by intravenous injection of ZDS-NPs in mouse. After 10 minutes, the hydrodynamic
diameter showed only a small increase as observed by SEC.
Gao at al20
employed ZDS to coat ultra-small iron oxide NPs, which they claim contain gadolinium
species (e.g., Gd2O3 nanoclusters), that had a good performance as T1 contrast agents. These novel
NPs have a constant hydrodynamic diameter of approximately 5 nm measured in PBS buffer and
FBS solution, again showing a low nonspecific protein interaction and a sufficiently long blood
half-life (ca 50 min). Thanks to their size, such NPs are rapidly filtered by kidneys and can be
passive targeted in tumors by the EPR effect, as tested in a human ovarian cancer as model.
Basing on these promising results about the potential application of ZDS-NPs in biomedical field,
we have decided to use in this PhD thesis the ZDS molecule to produce water-soluble iron oxide
NPs and we have compared them with a batch of NPs covered by the commercially available 2-[2-
(2-methoxyethoxy)ethoxy]acetic acid (MEEA)21
, with structure MeO(CH2CH2O)2CH2COOH. The
latter is about as large as ZDS, featuring a short PEG-like chain with a carboxylic acid group able
to graft the NP surface as carboxylate anion22
. Both coatings were characterized in detail by Fourier
23
Transform Infrared (FTIR) spectroscopy and Thermal Gravimetric Analysis (TGA) and their
behavior towards protein adsorption was evaluated by Dynamic Light Scattering (DLS). Then, we
have assessed the NP interaction with cells investigating their cytotoxicity, internalization, and
intracellular destinyusing human liver carcinoma cells (HepG2) in view of their high phagocytic
activity23-24-25
. To study the NP localization inside the HepG2 cells, we prepared a new fluorescent
catechol ligand.
Since ZDS and MEEA have similar molecular length, they form a coating of similar thickness.
Using the same batch of monodisperse iron oxide NPs, we have obtained information about the
nanosystem surface chemistry which is the main factor, as explained in the introduction, to produce
“stealth NPs”, essential for successful theranostic agents.
02. Preparation of iron oxide NPs coated with zwitterions
a. Synthesis of magnetic nanoparticles
Since NP properties are strongly size dependent, we planned to use NP comprising a crystalline iron
oxide core with a very small size dispersion so that investigation of the surface chemistry is not
hampered by size inhomogeneous NPs. The nanocrystal size and morphology can be controlled by
various synthetic procedures as reported in literature26-27
. A general way to produce monodisperse
NCs is based on the high-temperature decomposition of metal complex in a high boiling solvent in
the presence of a surfactant (solvothermal synthesis)28
. In particular, two procedures are well
known. In the hot-injection process, the metal complex is instantaneously decomposed by quickly
injection inside a hot mixture of solvent and surfactant while in the heating-up process all reagents
are dissolved in the solvent at moderate temperature and then the homogeneous reaction mixture is
heat up to high temperature. The crystal growth control diversifies the two procedures. In both
methods, reaction temperature and reactant concentrations determine the size distribution of the
NPs. The most common surfactants employed in the solvothermal synthesis of NPs to stabilize
them and modulate their dimension and shape are long-chain molecules such as oleic acid and
oleylamine. Moreover, the most common metal precursor used in hot-injection are metal carbonyls
(e.g. iron pentacarbonyl) while for heating-up procedures metal acetylacetonates or oleates are
widely employed.
We obtained iron oxide NPs with diameter ca. 9 nm following a modification of a hot-injection
procedure described in literature29
(see Experimental Section). We modified another procedure to
synthesize NPs30
to produce larger NCs (d ≈ 12 nm) by changing the molar ratio between metal
24
precursor and surfactant (from 1:4 to 1:3) in 1-octadecene (ODE). In both cases we produced
monodisperse spherical iron oxide NPs coated with oleic acid (OlAc-NPs), as confirmed by TEM
images (see Fig 4 and 5).
Fig 4. Smaller (8.7 nm) OlAc-coated NPs. Left) TEM image from Ref 21. Right) Histogram of the
NP diameters measured by the software PEBBLES31
.
Fig 5. Larger (11.4 nm) OlAc-coated NPs. Left) TEM image. Right) Histogram of the NP diameters
measured by the software PEBBLES31
.
4 6 8 10 12 14 160
100
200
300
400
500
600
700
800
Equiv. Diameter (nm)
NP
counts
All loaded lists
Equiv. Diameter (nm)
mean: 8.7
median: 8.7
st.dev.: 0.4 (4.6%)
No. of NPs: 1020
7 8 9 10 11 12 13 14 15 160
50
100
150
200
250
300
350
Equiv. Diameter (nm)
NP
counts
Equiv. Diameter (nm)
mean: 11.4
median: 11.4
st.dev.: 0.6 (5.4%)
No. of NPs: 679
25
For smaller iron oxide NPs the median diameter was <d> = 8.7 nm with a diameter standard
deviation σd = 0.4 nm, witnessing a very low dispersion σd/<d> = 4.6%. For larger NPs the median
diameter was <d> = 11.4 nm, with σd = 0.6 nm, showing again a very narrow dispersion σd/<d> =
5.4%.
Since the as-synthesized NPs have small size and magnetite (Fe3O4) and maghemite (γ-Fe2O3) have
very similar electron diffraction pattern (Fig 6), we can at most conclude that our NPs are iron oxide
NPs with cubic spinel structure and stoichiometry intermediate between magnetite and maghemite
(Fe3–xO4–x, 0 < x < 1).
Fig 6 . Electron diffraction pattern of OlAc-NPs. All diffraction rings are indexed with
reference to the magnetite structure. Image from Ref 21.
220
400
440
333
511
311
222
26
b. Catecholic ligands
The catecholic ligands used as iron oxide NP surfactants, were synthesized in collaboration with Dr.
Carmelo Drago (CNR, Istituto di Chimica Biomolecolare, Catania).
The zwitterionic dopamine sulfonate ( ZDS) (Fig 7) was obtained following a modified two-step
pathway based on the previously reported procedure17 and described in detail in the Experimental
Section. Briefly, commercially available dopamine hydrochloride was deprotonated with aq.
ammonia and reacted with propansultone to give the sulfonic acid DS, which was isolated and
further reacted with a large excess of MeI in the presence of an inorganic base to obtain the desired
zwitterion ZDS.
NH3
+ Cl
-OH
OH
+ OS
O O
OH
OH
NH S
O
O
O-
OH
OH
N+
S
O
O
O-
CH3
CH3
aq NH3
EtOH, 50 °C
DMF, 50 °C
MeI
DS ZDS
Fig 7. Synthesis of zwitterionic dopamine sulfonate ZDS 21
.
Instead the fluorescent catecholic ligand (FCL), whose structure is depicted in Fig 8, was employed
to study the intracellular fate of iron oxide NPs after their internalization into HepG2 cells. It was
produced as reported in detail in the Experimental Section.
OH
OH
NHO
NH O
O O
NH
O
O
O OHOH
O
3
Fig 8. Chemical structure of fluorescent catecholic ligand (FCL). The position of the benzamido
moiety is uncertain (5 or 6) because we used commercially available 5(6)-carboxyfluorescein.
27
c. Preparation of water-soluble NPs via ligand exchange
procedure
To prepare water soluble iron oxide NPs, we employed the above described batches of
monodisperse OlAc-coated NPs and we resorted to ligand exchange procedures in order to replace
the pristine hydrophobic coating (oleic acid) with the desired hydrophilic one.
In particular, both MEEA- and ZDS-coated NPs were obtained following pathway modification of
the literature procedure17
. A similar process was used to produce the fluorescent NPs, which were
coated with a mixture of FCL and ZDS. The presence of ZDS is required to endow the fluorescent
NPs with good colloidal stability in water. The coating procedure is described in detail in the
Experimental Section.
MEEA-, ZDS- and FCL-NPs were obtained from OlAc-NPs with <d> = 8.7 nm (see Fig 9), while
ZDS-NPs were produced also from OlAc-NPs with <d> = 11.4 nm (see Fig 10). The latter were
employed in DLS experiments. A summary of the NP morphological parameters before and after
the exchange processes is reported in Table 1 and Table 2, respectively (see below).
Fig 9. TEM images (top) and diameter histograms (bottom) of ligand-exchanged NPs prepared
from 8.7 nm OlAc –NPs. Left) MEEA-NPS. Center) ZDS-NPs. Right) FCL-NPs.
4 5 6 7 8 9 10 11 12 13 140
50
100
150
200
Equiv. Diameter (nm)
NP
counts
All loaded lists
Equiv. Diameter (nm)
mean: 8.8
median: 8.8
st.dev.: 0.6 (7.3%)
No. of NPs: 311
5 6 7 8 9 10 11 12 130
50
100
150
200
250
300
350
400
Equiv. Diameter (nm)
NP
counts
All loaded lists
Equiv. Diameter (nm)
mean: 9.0
median: 9.0
st.dev.: 0.5 (5.2%)
No. of NPs: 747
2 4 6 8 10 12 14 160
50
100
150
200
250
Equiv. Diameter (nm)
NP
counts
Equiv. Diameter (nm)
mean: 8.2
median: 8.1
st.dev.: 0.8 (9.6%)
No. of NPs: 326
28
<d> (nm) σd (nm) σd/<d> (%)
OlAc-NPs 8.7 0.4 4.6
MEEA-NPs 8.8 0.6 7.3
ZDS-NPs 9.0 0.5 5.2
FCL-NPs 8.1 0.8 9.6
Table 1. Summary of the morphological parameters before and after the exchange process for NPs
prepared from 8.7 nm OlAc –NPs.
Fig 10. TEM image of ZDS-NPs coming from the batch of OlAc–NPs with diameter ca. 12 nm
(“larger” OlAc-NPs). TEM image (left) and diameter histogram (right) of ZDS-NPs prepared from
11.4 nm OlAc –NPs.
Morphological parameters <d> (nm) σd (nm) σd/<d> (%)
OlAc-NPs 11.4 0.6 5.4
ZDS-NPs 12.3 0.7 5.5
Table 2. Summary of the morphological parameters before and after the exchange process for ZDS-
NPs prepared from 11.4 nm OlAc–NPs .
6 8 10 12 14 16 180
50
100
150
200
250
300
350
400
450
Equiv. Diameter (nm)
Equiv. Diameter (nm)
mean: 12.3
median: 12.3
st.dev.: 0.7 (5.5%)
No. of NPs: 767
29
d. Characterization of NP coating
We studied the coating at the surface of the prepared NP types by Fourier-Transform Infrared
(FTIR) spectroscopy and Thermogravimetric Analysis (TGA). UV-VIS spectroscopy was also used
for fluorescent NPs.
d.01. FTIR spectra
The FTIR spectra of OlAc-, MEEA-, and ZDS-NPs are shown in Fig 11 (left) along with the
corresponding FTIR spectra of pure ligands (right).
COATED NPS PURE LIGANDS
Fig 11. FTIR spectra of OlAc- (a), MEEA- (b), and ZDS-coated (c) iron oxide NPs (left). FTIR
spectra of the pure ligands are shown in the right subpanels. Image from Ref 21.
In all NP spectra the peak at 590 cm−1
can be ascribed to the iron oxide core. In the spectrum of
OlAc-NPs, strong C-H (2954, 2919, 2850 cm–1
) and carboxylate stretching peaks (1550 and 1463
cm–1
) are clearly recognizable. Instead, the spectrum of MEEA-NPs displays stretching bands
related to C-H (weak; 2956, 2923, 2855 cm–1
), carboxylate (1634 and 1410 cm–1
) and C-O-C (1200
and 1100 cm–1
)22
typical of MEEA, as proof of evidence that the ligand exchange procedure was
Ab
so
rba
nce a)
Ab
so
rba
nce b)
1000200030004000
c)
Wavenumber / cm-1
Ab
so
rba
nce
Ab
so
rba
nce a)
Ab
so
rba
nce b)
1000200030004000
c)
Wavenumber / cm-1
Ab
so
rba
nce
30
effective. Finally, the FTIR spectrum of ZDS-NPs shows, in addition to the weak C-H stretching
peaks (2850-2950 cm–1
), several diagnostic bands such as the S-O stretching peak (1209 cm−1
), the
C-H deformation peaks of the dimethylammonium group (1496 and 1042 cm−1
), and the ring
stretching (1596 cm−1
) and C-H deformation (914-732 cm−1
) peaks of 1,2,4-trisubstituted
benzenes21
. The ZDS-NPs spectrum is in agreement with that reported in literature for iron oxide
NPs coated with a short siloxane sulfobetaine32
. The intensity of the peaks in the C-H stretching
region decreases passing from OlAc to MEEA and ZDS as a consequence of the efficient ligand
exchange procedure.
d.02. TGA curves
MEEA- and ZDS-NPs were subjected to Thermogravimetric Analysis (TGA) by heating the NPs in
air up to 950 °C. The TGA curves are shown in Fig 12.
Fig 12. TGA curves of MEEA- and ZDS-coated iron oxide NPs.
In the case of MEEA-NPs the weight loss is 16.5%, in agreement with that already reported in
literature22
. Such percentage corresponds to a coating density of 6 molecule/nm2 of MEEA in the
coating, similar to the published data of 4.2-4.7 molecule/nm2
evaluated for aliphatic carboxylic
acids having comparable size33
. Instead, TGA curves of ZDS-NPs show two percentage weight
losses: 33% in the 100-440°C range and 12% in the 440-580°C range. We attributed the first weight
loss to the decomposition of the aliphatic part of the ZDS and the second one to the combustion of
the aromatic part, also considering that the aliphatic:aromatic weight ratio in ZDS is about 3:1. In
31
this case, the total weight loss (45%) corresponds to a very high coating density of 15 molecule/nm2
that we have attributed to the presence of a ZDS bilayer at the NP surface, as already observed in
the literature for zwitterionic molecules adsorbed onto the oleic acid coating of NPs 16 (i.e. with no
ligand replacement).
d.03. UV-Vis spectra
The fluorescent coating of iron oxide NPs, comprising a mixture of ZDS and FCL was analyzed by
UV-Visible spectroscopy (Fig 13). After purification by dialysis to remove the free ligands, we
registered the absorption spectrum of ZDS/FLC NPs. As visualized in Fig 13, it was in agreement
with that of the 5(6)-carboxyfluorescein chromophore of FLC, supporting the successful
achievement of the desired fluorescent coating. Moreover, from the quantitative analysis of the UV-
Vis spectrum we evaluated that the number of FCL molecules grafted to one iron oxide NP is
around 4∙102. This value can be compared with the ca. 3∙10
3 anchored ZDS molecules per NP
estimated from the above TGA results.
Fig 13. UV-Visible spectrum of fluorescent NPs coated with a mixture of ZDS and FLC in water
(solid line) compared with that of 5(6)-carboxyfluorescein (dotted line, shifted upwards by 0.012
absorbance units for the sake of clarity). Image from Ref 21.
32
03. Study of NP colloidal stability
We decided to employ Dynamic Light Scattering (DLS) to study the colloidal stability of MEEA-
and ZDS-NPs, first in deionized (DI) water and then in the complete culture medium of HepG2
cells. We have analyzed both the intensity weighted size distribution PI and the volume-weighted
size distribution PV.
In deionized water, as shown in Fig 14, both PI and PV for MEEA-coated NPs display a single peak
with mean diameter DI ≅ DV ≅ 750 nm. Therefore, these NPs were present in DI water as
aggregates. Instead, ZDS-coated NPs were well dispersed in DI water, displaying a major (85%)
peak in PI at DI = 23 nm and a minor (15%) peak at DI = 240 nm. The size related to the major
(99%) peak in the volume weighted distribution (DV = 12 nm) is in agreement with values reported
in literature19
and confirms that the ZDS layer is less than 2 nm thick. Only a minor fraction of
ZDS-NP mass is present as aggregates as evidenced by the peak area ratio of both PI (85:15) and PV
(99:1).
Fig 14. Intensity-weighted diameter distribution PI (left) and volume-weighted diameter distribution
Pv (right) of dispersions of MEEA- (red) and ZDS-coated (black) NPs in deionized water; PI and Pv
of the cell culture medium (green) is also shown. Images from Ref 21.
The ζ potential of ZDS-coated NPs in DI water was measured. Despite that ZDS is globally neutral
molecule, the ζ potential of ZDS-NPs in DI was negative, ζ = −8.6 mV, in conformity with that
previously measured for quantum dots coated with a polymer exhibiting sulfobetaine pendant
moieties (−13.1 mV)34
and Au NPs covered with sulfobetaine zwitterionic surfactants (−17.9/−14.8
mV)2.
The DLS results provide an explanation of the very different visual appearance of water dispersion
of MEEA- and ZDS-NPs having similar NP concentration (0.15 - 0.20 mg Fe/ml). Indeed, fresh
33
dispersions of MEEA-NPs appeared as turbid since they formed aggregates, while ZDS-NPs gave
dark, clear, stable solutions (Fig 15).
Fig 15. Water dispersions of MEEA- (A) and ZDS-coated (B) iron oxide NPs. Image from Ref 21.
Since ZDS-NPs appeared as well dispersible in DI water, we decided to explore their stability in
both the Roswell Park Memorial Institute (RPMI) cell culture medium (which is serum free) and the
complete cell culture medium (RPMI supplemented with 2 mM L-glutamine, 100U/ml antibiotic, 1
mM sodium pyruvate and 10% FBS), which will be used to grow the HepG2 cells for the in vitro
experiments described below. To this purpose, we analyzed the DLS intensity and size distribution
of NP dispersions at the concentration and for the time interval used in the uptake experiments,
starting from 9 nm ZDS-NPs.
The scattered intensity Is is very sensitive to an increase of NP size due to protein adsorption or
aggregation since Is is proportional to the sixth power of the NP size (e.g., a 10% size increase
causes a 2.5-fold increase of Is). As shown in Fig 16, the scattered intensity Is was constant for 24 h
in the case of the complete cell culture medium while Is displays a minor increase only at long
times, especially in the 21−24 h range, for the dispersion of ZDS-NPs in the complete cell culture
medium. These data witness the good colloidal stability of our dispersions and the absence of
significant protein adsorption by ZDS-NPS. These results are confirmed considering that the
volume-weighted size distribution PV did not display significant variations over 24 h (Fig 16).
34
Fig 16. Comparison of DLS of complete cell culture medium with (blue) and without (green) ZDS-
NPs. Left) intensity of the scattered light Is as a function of time t (the inset shows the initial
behavior). Right) Volume weighted distribution PV of the hydrodynamic diameter. Circles: t = 0 h,
triangles t = 24 h. Images from Ref 21.
The first order autocorrelogram g1(τ) and intensity-weighted size distribution PI (Fig. 17) indicate
that PI increased after 24 h. However, we have to consider that both Is and PI are very sensitive to
size changes (Is, PI ∝ D6), so the size increase occurring after 24 h can be considered negligible.
Indeed, this variation can be ascribed to changes occurring in the medium during time, since both
g1(τ) and PI curves have similar behavior. For this reason, we could exclude that protein adsorption
at the ZDS-NP surface occurred. Note that this process normally develops in a few minutes after
contacting NPs and proteins and leads to a huge increase in Is35
.
Fig 17. Normalized first-order autocorrelograms g1(τ) (left) and intensity-weighted distribution PI
(right) of ZDS-NPs in complete cell culture medium (blue) and of the pure cell culture medium
(green). Circles or triangles denote data at 0 h or at 24 h. Images from Ref 21.
35
To confirm the good stability of ZDS-coated NPs, we carried out the same DLS experiments
employing ZDS-coated iron oxide NPs, having a diameter ca. 12 nm. Their DV in deionized water
was equal to 15 nm. After dispersing the NPs (0.05 mg/ml) in RPMI serum-free, we measured DI =
19−23 nm and DV = 15 nm. The NPs were colloidally stable for 24 h and a very minor aggregation
was observed in PI (Fig 18).
a)
b)
c)
Fig 18. DLS of 12 nm ZDS-NPs in RPMI medium (serum free) at 0 (black) and 24 h (red). a) First-
order autocorrelation function g1(τ), b) intensity-weighted diameter distribution PI, c) volume-
weighted diameter distribution PV. Image from Ref 21.
Next, DLS experiments were carried out on dispersions of 12 nm ZDS-coated NPs in the complete
cell culture medium (Fig 19). Only very minor size changes (and hence protein adsorption) were
observed.
36
a)
b)
c)
Fig 19. DLS of 12 nm ZDS-coated iron oxide NPs in cell culture medium (modified RPMI + 10%
FBS) at 0 (black) and 24 h (red). a) Normalized first-order autocorrelation function g1(τ), b)
intensity-weighted diameter distribution PI, c) volume-weighted diameter distribution PV. Image
from Ref 21.
Finally we studied the behavior of 12 nm ZDS-coated NPs in PBS 1× added with 10% FBS. All
results confirmed the absence of protein adsorption and the substantial colloidal stability after 24 h
for these nanosystems (Fig 20).
37
a)
b)
c)
Fig 20. DLS of 12 nm ZDS-coated iron oxide NPs in PBS (1x) added with 10% FBS at 0 (black) and
24 h (red). a) Normalized first-order autocorrelation function g1(τ), b) intensity-weighted diameter
distribution PI, c) volume-weighted diameter distribution PV. Image from Ref 21.
04. NP Cytotoxicity
All biological experiments were made in collaboration with the group of Dr. Bice Chini (CNR,
Istituto di Neuroscienze, Milano).
We tested the cytotoxicity of MEEA- and ZDS-coated iron oxide NPs by the MTS test, a
colorimetric method that quantifies the metabolic activity of cells by evaluating the reduction of [3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS)
(CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). Viable cells reduce
MTS molecule by NAD(P)H-dependent dehydrogenase enzymes to generate a colored formazan
product that is soluble in the cell culture medium and whose absorbance can be measured at 490-
500 nm. The applied procedure is described in detail in the Experimental Section.
We evaluated the toxic effect on HepG2 cells of iron oxide NPs (added to the cell medium to final
concentrations of 2.5, 5, 10, 25, 50, and 100 µg Fe/ml) after 24 h exposure. The percentage
38
difference between treated and untreated cells was employed to express cell viability. In general,
we observed that the addition of NPs to the cell culture medium did not significantly perturb its pH
and osmolarity. In more detail, we found that the highest doses of MEEA-coated NPs (50 and 100
µg Fe/ml) induced a small cytotoxic effect whereas even the highest dose of ZDS-NPs did not
induce a cytotoxic effect (Fig 21). Iron does not interfere with the MTS test even at the highest NP
concentration employed in these experiments (100 μg/ml)36
.
Fig 21. Cell viability upon exposure to increasing concentrations of MEEA- (left) or ZDS- coated
(right) NPs [MTS cell viability assay]. Obtained data are presented as mean ± SEM and compared
to control values using ANOVA test. * p<0.05; ***p<0.001. Image from Ref 21.
05. Uptake experiments by HepG2 cells
As already mentioned in the Introduction, we employed human liver carcinoma cells (HepG2) for
the NP uptake experiments since these cells display a high phagocytic activity. We evaluated the
NP internalization in two ways: qualitatively, by Prussian Blue staining and imaging, and
quantitatively, by UV-Vis spectroscopy. In our assays, HepG2 cells were treated for 24 h with NPs
added to the culture medium at final concentration of 0, 2.5, 10, 25, 50 or 100 µg Fe/ml.
e. Qualitative iron evaluation
To carry out a qualitative investigation of the NP internalization, HepG2 cells were exposed to
increasing concentration of iron oxide NPs and then the internalization was observed by Prussian
Blue staining of the washed cells. The complete procedure is described in detail in the Experimental
Section. For both MEEA-NPs and ZDS-NPs the intracellular uptake increases with the NP dose.
39
Prussian Blue staining clearly revealed much larger intracellular MEEA-NPs than ZDS-NPs (Fig
22), probably because MEEA-NP aggregates were internalized.
Fig 22. Prussian Blue staining of HepG2 cells after treatment with iron oxide NPs. HepG2 cells
were exposed for 24 hours to different concentrations of MEEA- (top) or ZDS-coated (bottom) NPs,
washed and processed for Prussian Blue staining and optical imaging. Black aggregates indicate
the presence of iron. Scale bar = 10 μm. Image from Ref 21.
f. Quantitative iron quantification
HepG2 cells were treated for 24 h with NPs added to the culture medium at final concentration of 0,
2.5, 10, 25, 50 or 100 µg Fe/ml. To quantify the intracellular iron content we used a procedure
described in detail in the Experimental Section. In brief, cellular pellets were digested with
concentrated HCl/HNO3 and the resulting aqueous solution was treated with tiron (disodium 4,5-
dihydroxybenzene-1,3-disulfonate) at pH ≈ 7 to form the red complex Fe(tiron)3. This complex is
suitable for quantification by UV-Vis spectroscopy37
. UV-Vis data were analyzed by fitting a broad
spectral interval (400-800 nm), thus ensuring better accuracy with respect to previous methods38,39
.
The cell protein content was quantified by the BIORAD-Dc protein assay in order to express the NP
internalization as mass(internalized iron)/mass(protein). The native iron content of HepG2 cells was
(mFe/mprotein)control = (0.4 ± 0.1) μgFe/mgprotein. The iron uptake was calculated as U =
(mFe/mprotein)treated – (mFe/mprotein)control, where mFe and mprotein are the cellular iron and protein mass,
respectively, both evaluated after 24 h treatment and averaged over the replicates (Fig 23).
40
Fig 23. NP internalization in HepG2 cells after 24 h incubation with increasing concentrations of
MEEA- (left) or ZDS-coated (right) NPs. Note the different vertical scales in the subpanels. Results
are presented as mean ± SEM and their significance was assessed using Student’s t-test, after
testing for equal variance using the F-test. Student’s t-test was applied to test for uptake differences
between adjacent doses (*) and with respect to the control (#).* p<0.05; # p<0.05, ## p<0.005.
Image from Ref 21.
The plots show that the NP internalization depends on the dose of NPs administered to HepG2 cells
in the culture medium for both MEEA- and ZDS-coated iron oxide NPs. Experimental constraints,
for instance an excessive volume of NP dispersion added to the culture medium, did not allow us to
increase the NP final concentration to more than 100 µg Fe/ml. According with Prussian Blue
staining observations, the amount of NPs taken up by HepG2 cells was closely related to the
surfactant type: in particular the amount of internalized MEEA-NPs was 20-30 times higher than
that of ZDS-NPs. Since these NPs have the same core and coating size, the surface chemistry is the
only responsible for their different cell internalization. We could ascribe these results to the
dissimilar surfactant charge (zwitterionic vs. neutral), which might influence the interaction of NPs
with cell membranes. However, DLS results showed that such chemical differences primarily
influenced NP colloidal stability in the culture medium, so that MEEA-coated NPs were
internalized to a larger extent probably thanks to their presence as micrometer-sized aggregates35
,
maybe involving a size-dependent uptake mechanism. On the other hand, DLS showed that ZDS-
NPs did not vary their hydrodynamic size owing to protein adsorption or aggregation in the full cell
culture medium, so they interacted as well-dispersed with cells affording low internalization.
We compared our uptake data with those reported in literature. First, we considered internalization
of iron oxide NPs by HepG2 cells. In the case of MEEA-coated NPs our results are higher but
comparable with the internalization observed for NPs forming aggregates. Some examples are the
uptake of cationic and galactose-decorated magnetoliposomes40
, iron oxide NPs covered with
41
glucosamic acid41
or aminopropylsiloxane groups24
and commercially available “anionic” NPs42
.
Considering NPs well-dispersed to compare their behavior with ZDS-coated NPs, nanocrystals
covered with PEG600 carboxylic acid24
and anionic magnetoliposomes40
were internalized in a
larger amount with respect to our ZDS nanosystems. A slighty lower uptake than that of ZDS-NPs
was reported for iron oxide NPs coated with dimercaptosuccinic acid23
. Second, we considered two
reports concerning the uptake of NPs coated with zwitterions, in particular with a polymeric coating
functionalized with carboxybetaines. One report described the internalization by RAW 264.7 cells
of well dispersed NPs (D = 19 nm) coated with polyacrylic acid bearing ammonium groups. The
second report14
described the exposure of RAW 264.7 and also HUVEC cells to NPs (D ≈ 130 nm)
coated with a polycarboxybetainemethacrylate polymer. Assuming that HepG2, RAW 264.7 and
HUVEC cells have a comparable protein content, the internalizations of NPs coated with a
zwitterionic polymer and with our small ZDS molecule were very similar. These comparisons
suggest that the hydrodynamic diameter of zwitterionic NPs scarcely affects their uptake by cells.
06. Study of the intracellular NP fate
Finally, to explore the intracellular destiny of ZDS-NPs, we used fluorescent iron oxide NPs coated
with a mixture of ZDS and FCL. TGA and UV-Vis data showed that the number of FCL molecules
in the coating is about 10% of the number of ZDS molecules. HepG2 cells were exposed for 24
hours to different concentrations of the fluorescent NPs, then washed and processed for optical
imaging according to the procedure described in detail in the Experimental Section. First, confocal
microscopy images again witnessed a dose-dependent uptake by cells (Fig 24). Moreover, these
NPs did not stain the plasma membrane, a remark supporting that the final washing of HepG2 with
PBS solution was effective in removing non-internalized NPs.
42
Fig 24. Confocal images of HepG2 cells treated with fluorescent NPs. The NPs are coated with a
mixture of ZDS and FCL ca. 9:1 mol/mol. Scale bar = 20 μm. Inset scale bar = 10 μm. Image from
Ref 21.
However, the most important information deriving from the study of the internalization of
fluorescent NPs concerned the intracellular fate of the NPs (green) which was studied by labeling in
vivo lysosomes with Lysotracker-Red (red) and cell nuclei with DAPI (blue). Co-localization of the
green and red signals, 6 or 24 hours after the addition of NPs in the cell medium, proved that these
NPs in HepG2 cells were localized in lysosomes (Fig 25).
43
Fig 25. Lysosomal accumulation of fluorescent NPs, coated with a mixture of ZDS and FCL, in
HepG2 cells. Cells were exposed to these NPs for 6 or 24 hours. Arrowheads indicates sites of
colocalization between the green (NPs) and the red signal (Lysotracker). Scale bar: 5 μm. Image
from Ref 21.
Such result could be interesting for the potential application of ZDs-coated NPs in cancer therapy
because magnetic NPs with the same lysosomial destiny39-43
have already been reported in the
literature as efficient killers of tumor cells. Indeed, they induced cell death through the release of
the lysosome content in the cytosol when exposed to an external magnetic field43
.
44
07. Conclusions
In this part of my PhD thesis, we prepared iron oxide NPs coated with a small zwitterionic
molecule, ZDS, and we characterized them from the physico-chemical point of view, analyzing
their coating structure and their colloidal stability in order to explore the protein adsorption on the
NP surface. On the basis of such results, we also explored their cellular toxicity, uptake and
intracellular destiny by HepG2 cells.
We compared the ZDS-coated NPS with MEEA-coated NPs obtained from the same batch of
nanocrystals, with the aim of investigating the effects due to the different surface chemistry. ZDS-
coated NPs have a high density of sulfobetaine ligands, which provides NPs with high colloidal
stability even in the complete cell culture medium by avoiding NP aggregation or precipitation and
the protein adsorption at their surface. Conversely, MEEA-coated NPs show low colloidal stability
in deionized water and the fast formation of micrometer-sized aggregates.
Cellular toxicity and the efficiency of aspecific uptake are affected by such different behavior,
which has a deep impact on the in vitro interaction between NPs and cells. Therefore, we have
confirmed the centrality of NP surface chemistry in NP-cell interactions and why it is important to
monitor the NP dispersion during biological in vitro assays.
ZDS-NPs were not toxic for HepG2 cells even at the highest administered concentration (100 µg
Fe/ml), while MEEA-NPs caused cytotoxicity already at 50 µg Fe/ml. Both NP types are
aspecifically internalized in HepG2 cells in a dose-dependent way, but to a very low extent for the
zwitterionic NPs. One could benefit from this aspect when highly selective targeting through
specific NP functionalization is desired.
Finally, ZDS-coated NPs showed a lysosomal intracellular destiny, which is desirable when we
want to destroy targeted cells, such as in tumor treatment. Taken together, all the advantages
underlined for ZDS-coated NPs, indicate that they could be considered as a good backbone for the
development of theranostic nanosystems.
45
08. Experimental section
g. Materials
All chemicals [dopamine hydrochloride (98.5%), 1,3-propansultone, iodomethane, 4,7,10-trioxa-
1,13-tridecanediamine, triethylamine, trityl chloride, diglycolic anhydride, pyridine (Pyr), N-
hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N-
diisopropylethylamine (DIPEA), triisopropylsilane (TIPS), trifluoroacetic acid (TFA), N-
hydroxysuccinimide (NHS), N,N'-diisopropylcarbodiimide (DIC), iron pentacarbonyl (Fe(CO)5),
oleic acid (OlAc, 90%), octyl ether (99%) , 2-[2-(2-methoxyethoxy)ethoxy] acetic acid (MEEA,
90%)] and solvents [methanol, ethanol (98%), octadecene (ODE, 90%), aqueous ammonia,
dimethylformamide (DMF), acetone (tech. grade), ethyl acetate, dichloromethane (DCM),
diisopropylether] were acquired from Sigma-Aldrich and employed as received without further
purification. To digest NPs we used concentrated acids (HNO3 and HCl) that were Aldrich Trace
Select reagents. Deionized (DI) water was obtained using a Milli-Q Plus water purification system
(resistivity > 18.2 mΩ cm at 25 °C). The human liver carcinoma cells (HepG2) were obtained from
the American Type Culture Collection.
h. Procedures
h.01. Acronyms used in the experimental section
OlAc = oleic acid
Fe(CO)5 = iron pentacarbonyl
RT = room temperature
DMF = dimethylformamide
D2O = deuterium oxide
TEA = triethylamine
NaHCO3 = sodium hydrogen carbonate
46
Na2SO4 = sodium sulfate
CDCl3 = deuterochloroform
TrCl = trityl chloride
DCM = dichloromethane
EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
Pyr = pyridine
HOBt = N-hydroxybenzotriazole
DIPEA = N,N-diisopropylethylamine
CH3OH/ MeOH = methanol
iPrOH = isopropanol
CD3OD = deuterated methanol
TIPS = triisopropylsilane
TFA = trifluoroacetic acid
i-Pr2O = isopropyl ether
NHS = N-hydroxysuccinimide
DIC = N,N'-diisopropylcarbodiimide
CH3CN = acetonitrile
DI water = deionized water
PBS = phosphate buffer saline
PFA = paraformaldehyde
HCl = hydrochloric acid
HNO3 = nitric acid
DAPI = 4′,6-diamidine-2′-phenylindole dihydrochloride
tiron = disodium 4,5-dihydroxy-1,3-benzenedisulfonate
h.02. Preparation of 9 nm iron oxide NPs
9 nm iron oxide NPs were produced according to a modification of a published literature
procedure29
. Oleic acid (OlAc, 600 l, 1.89 mmol) was dispersed in octyl ether (10 ml) and oxygen
was repeatedly removed from the solution at RT by vacuum/nitrogen cycles. Afterwards, the
solution was heated to 105 °C under nitrogen and, after another degassing cycle, Fe(CO)5 (200 l,
1.52 mmol) was injected into the reaction mixture (Fe(CO)5:OlAc = 1:1.25 mol/mol). The mixture
was heated to reflux (285 °C) with a rate of 3.3 °C/min and maintained at this temperature for 25
47
min. Around 260 °C the color of the reaction mixture changed from pale yellow to black. The
reaction crude, cooled at RT, was treated with acetone and the precipitated NPs were collected by
centrifugation (6000 rpm for 10 min). The NPs were washed several times with acetone or ethanol
and recovered by centrifugation (6000 rpm for 10 min). Finally, the OlAc-coated NPs were
dispersed in hexane (20 ml).
h.03. Preparation of 12 nm iron oxide NPs
12 nm iron oxide NPs were produced according to a modification of a published literature
procedure30
. Fe(CO)5 was injected in a solution of 1-octadecene and oleic acid (Fe(CO)5:OlAc = 1:3
mol/mol) at 120 °C and then heated to 320°C for 3 h under nitrogen. After cooling at RT, the NPs
were precipitated by adding acetone and collected by centrifugation. Then, they were repeatedly
washed with acetone and dispersed in toluene.
h.04. Synthesis of zwitterionic dopamine sulfonate (ZDS)
ZDS molecule was synthesized in two steps following the Scheme 1, a modification of an already
reported procedure17
, which is detailed below.
NH3
+ Cl
-OH
OH
+ OS
O O
OH
OH
NH S
O
O
O-
OH
OH
N+
S
O
O
O-
CH3
CH3
aq NH3
EtOH, 50 °C
DMF, 50 °C
MeI
DS ZDS
Scheme 1. Synthesis of ZDS molecule 21
.
48
h.04. 1. Synthesis of dopamine sulfonate, DS
28% aq. ammonia (416 μL, 3.00 mmol) and 1,3-propanesultone (799 mg, 6.50 mmol) were slowly
added to a solution of dopamine hydrochloride (1.14 g, 6.0 mmol) in ethanol (150 mL) under inert
gas. The mixture was warmed up to 50 °C and stirred for 72 h, giving a white precipitate. The solid
(DS) was recovered by centrifugation, washed with ethanol (3 x 10 mL) and finally dried under
reduced pressure.
1H-NMR (400 MHz, D2O): δ (ppm) 2.00 (m, 2H), 2.78-2.82 (m, 2H), 2.86-2.91 (m, 2H), 3.06-3.12
(m, 2H), 3.16-3.18 (m, 2H), 6.72-6.74 (m, 1H), 6.78- 6.82 (m, 2H). 13
C-NMR (100 MHz, D2O): δ
(ppm) 21.1, 30.9, 46.1, 47.8, 48.6, 116.4, 121.1, 128.9, 143.0, 144.2.
h.04. 2. Synthesis of zwitterionic dopamine sulfonate, ZDS
Anhydrous sodium carbonate (0.254 g, 2.40 mmol) was added to a solution of DS (0.329 g, 1.00
mmol) in DMF (150 mL) and mixed under nitrogen for 2 h. After that, iodomethane (2.2 mL, 35
mmol) was added to the mixture, which was heated to 50 °C and continuously stirred overnight.
After reaction completion, the solvent was evaporated under reduced pressure, obtaining an oil
which was purified through several consecutive crystallization steps with DMF/ethyl acetate (1:10
v/v, 50 mL) finally leading to a white solid collected by centrifugation and dried under reduced
pressure.
1H-NMR (400 MHz, D2O): δ (ppm) 2.21 (m, 2H), 2.92-2.95 (m, 4H), 3.13 (s, 6H), 3.47-3.51 (m,
4H), 6.74-6.76 (m, 1H), 6.83-6.88 (m, 2H). 13
C-NMR (400 MHz, D2O): δ (ppm) 18.1, 27.7, 47.1,
50.7, 62.0, 64.6, 116.5, 121.2, 128.2, 143.0, 144.2.
h.05. Synthesis of fluorescent catecholic ligand ( FCL)
The fluorescent catecholic ligand (FCL) was produced following the Scheme 2 reported below
starting from the product commercially acquirable 4,7,10-trioxa-1,13-tridecanediamine (1).
49
OH
OH
NHO
NH O
O O
NH
O
O
O OHOH
O
3
NH2 O NH23
1
NH2 O NH
Ph
Ph
Ph3
2
NH2 O NH
Ph
Ph
Ph3
3
O
O
OO
NH2
OH
OH
3
4
NH O NH
Ph
Ph
Ph
O
ONH
O
OH
OH
3
5
NH O NH2
O
ONH
O
OH
OH
6
O
O OHOH
O
OH
O
Scheme 2. Synthetic pathway leading to the FCL (6)21
.
50
h.05.1 Synthesis of compound 2
A solution of compound 1 (5 g, 22.70 mmol) in dichloromethane (250 ml) and TEA (0.475 mL,
3.41 mmol) was stirred for 30 min. After cooling to 0°C with an ice-bath, trityl chloride (TrCl, 632
mg, 2.27 mmol) was added to the crude in small portions. Then the ice-bath was removed and the
reaction mixture was maintained overnight at RT. The DCM phase was washed twice with a
NaHCO3 saturated solution, dried with anhydrous Na2SO4, separated out and finally evaporated
under reduced pressure to give product 2 (1.07 g, 2.27 mmol, quantitative yield).
1H-NMR (400 MHz, CDCl3, 25 °C): δ = 7.43-7.37 (m, 6 H, aromatic protons Tr), 7.23-7.17 (m, 6
H, aromatic protons Tr), 7.13-7.08 (m, 3 H, aromatic protons Tr), 3.58-3.41 (m, 12 H,
CH2OCH2CH2OCH2CH2OCH2), 2.78 ( t, 2 H, CH2NH2 ), 2.20-2.11 (m, 2 H, CH2NH ) 1.76-1.63
(m, 4 H, CH2CH2NH2, CH2CH2NH). ESI-MS: m/z 463.5 [M+H]+.
h.05.2 Synthesis of compound 3
Compound 2 was dissolved in dry DMF (10 ml); then diglycolic anhydride (527 mg, 4.54 mmol)
and pyridine (Pyr, 0.736 mL, 9.08 mmol) were added to the solution and were mixed for 16 h at RT.
The crude was acidified to pH 5 with an aqueous solution of 5% citric acid. The desired and
practically pure compound 3 was extracted from water with DCM, dried on anhydrous Na2SO4,
filtered off and finally obtained by evaporation under reduced pressure.
1H-NMR (400 MHz, CDCl3, 25 °C): δ = 7.63-7.51 (m, 5 H, aromatic protons Tr), 7.44-7.31(m, 10
H, aromatic protons Tr), 4.19 (s, 2H, CH2COOH), 4.13(s, 2H, OCH2CO), 3.55(m, 6 H), 3.52(t, 2
H,CH2NHCO), 3.45-3.35(m, 6 H), 3.2(t, 2 H, CH2NHCTr), 2.04(t, 2 H, CH2CH2NHCTr), 1.79(t, 2
H, CH2CH2NHCO). 13
C NMR (400 MHz, CDCl3, 25 °C): δ = 138.8, 128.8, 128.7, 128.6, 71.3,
70.6, 70.5, 70.3, 70.1, 69.9, 69.4, 69.2, 45.4, 36.9, 36.3, 31.3, 28.7, 25.4 .ESI-MS: m/z 579.5
[M+H]+.
h.05.3 Synthesis of compound 4
EDC (154 mg, 0.801 mmol), HOBt (108 mg, 0.801 mmol) and DIPEA (0.458 mL, 2.676 mmol)
were consecutively added at RT to a mixed solution of compound 3 (426 mg, 0.736 mmol) in DMF
(5mL). After complete dissolution of the reagents, dopamine hydrochloride (127 mg, 0.669 mmol)
was added and the reaction was stirred until completion, as assessed by HPLC-MS. Finally, the
DMF was evaporated in vacuo and the crude product was recovered as pure 4 by BiotageTM C18
51
reverse phase chromatography. We applied the following BiotageTM eluant conditions: gradient
from 90% H2O-10% CH3OH-iPrOH(6:4) to 100% CH3OH-iPrOH (6:4). 286 mg, 0.399 mmol, 60%
yield.
1H-NMR (400 MHz, CD3OD, 25 °C): δ = 7.46-7.40 (m, 6 H, aromatic protons Tr),7.29-7.21 (m, 6
H, aromatic protons Tr), 7.20-7.14 (m, 3 H, aromatic protons Tr), 6.68 (d, 1 H, catechol, Jortho= 8.3
Hz), 6.65 (d, 1 H, catechol, Jmeta= 1.7 Hz), 6.52 (dd, 1 H, catechol, Jortho= 8.3 Hz, Jmeta= 1.7
Hz), 4.00 (s, 2 H), 3.98 (s, 2 H), 3.61-3.51 (m, 8 H), 3.50-3.44 (m, 4 H), 3.41 (t, 2 H), 2.67 (t, 2 H),
2.25 (t, 2 H), 1.76 (qt, 4 H), 1.15 (d, 2 H). 13
C NMR (400 MHz, CDCl3, 25 °C): δ = 129.7, 128.4,
127.0, 120.8, 116.6, 116.1, 71.3, 71.26, 71.22, 71.06, 70.99, 70.93, 68.6, 41.3, 40.5, 36.3, 34.5,
29.8, 29.0. ESI-MS: m/z 714.5 [M+H]+.
h.05.4 Synthesis of compound 5
A solution of compound 4 (120 mg, 0.168 mmol) solved in DCM (5 mL), TIPS (0.070 mL, 0.336
mmol) and TFA (0.250 mL) were stirred for 2 h at RT. Then, DCM was evaporated under reduced
pressure. The crude residue was treated with water and extracted twice with i-Pr2O. The organic
phase was then dried on Na2SO4, removed in vacuo affording the product 5 (95 mg, yield 82%)
which can be used without further purification. 1H-NMR (400 MHz, D2O, 25 °C): δ 6.90 (d, 1 H,
catechol, Jortho= 7.6 Hz) 6.84 (d, 1 H, catechol, Jmeta= 1.76 Hz), 6.75 (dd, 1 H, catechol, Jortho = 7.6
Hz, Jmeta = 1.76 Hz), 4.10 (s, 2 H), 4.03 (s, 2 H), 3.78-3.66 (m, 10 H), 3.62 (t, 2H), 3.53 (t, 2H), 3.30
(t, 2 H), 3.15 (t, 2 H), 2.78 (t, 2 H), 1.99 (qt, 2 H), 1.84 (qt, 2 H). ESI-MS: m/z 472.3 [M+H]+.
h.05.5 Synthesis of compound 6 (FCL)
NHS (30 mg, 0.225 mmol) and DIC (90 μl, 0.56 mmol) were consequently added to a mixture of
5(6)-carboxyfluorescein (85 mg, 0.225 mmol) in dry DMF (4 ml) and then were stirred at RT for 18
h. After reaction completion, DMF was removed under reduced pressure and the residue was left
under the high vacuum during the night. Then, it was diluted in dry DMF (3 ml) and then slowly
added to a solution of compound 5 (0.225 mmol) and DIPEA (154 μl, 0.90 mmol) in dry DMF (2
ml), that was magnetically mixed over night at RT. Finally the organic solvent was evaporated and
compound 6 was purified by HPLC using a C18 reverse column. We applied the following HPLC
52
eluant conditions: from 90% H2O/ 10% CH3CN to 100% CH3CN with a flow rate 20 ml/min. Yield
of pure FCL: 57% (106 mg, 0.128 mmol).
1H-NMR (400 MHz, D2O, 25 °C): δ = 8.40 [s, 1H, 5(6)-carboxyfluorescein], 8.14 [dd, 1H, 5(6)-
carboxyfluorescein], 8.06 [dd, 1H, 5(6)-carboxyfluorescein], 7.61 [s, 1H, 5(6)-carboxyfluorescein],
7.26 [d, 1H, 5(6)-carboxyfluorescein], 6.72-6.43 (m, 10H, 5(6)-carboxyfluorescein and catechol),
3.94- 3.95 (2s, 4H), 3.57- 3.23 (m, 18H), 2.58-2.62( t, 2H), 1.89-1.60 (m, 4 H). 13
C NMR (400
MHz, D2O, 25 °C): δ = 133.75, 129.32, 129.18, 129.06, 119.67, 115.50, 115.03, 102.24, 70.11,
70.09, 70.05, 69.88, 69.79, 58.60, 48.48, 40.47, 34.45, 28.97. ESI-MS: m/z 830.7 [M]+; 852.7
[M+Na]+.
h.06. Ligand exchange procedures
h.06.1 Preparation of MEEA- or ZDS-coated NPs
The oleic acid coating of NPs was displaced with either MEEA or ZDS by a two-step ligand
exchange procedure17
.
First as-synthesized OlAc-NPs (17 mg) were gently stirred at 70 °C for 5 h in the presence of a
solution of MEEA (327 μl) in methanol (7.5 ml). NPs were collected by centrifugation after the
addition of acetone and hexane in succession.
In the second step, in a mixture of DMF and water (7:1), the MEEA-coated NPS were treated with
ZDS (250 mg, to obtain ZDS- NPS) or MEEA again (243 mg, to improve the coating density of
MEEA- NPs) and heated up to 70°C for 12 h. The resulting NPs were precipitated with acetone and
were solved in deionized water. Finally they were purified from the excess of surfactant by dialysis
(MWCO 12500 Da) at different times: 48 h for ZDS-NPs, 3 hours for MEEA-NPs.
h.06.2 Preparation of FCL-coated NPs
Fluorescent iron oxide NPs was obtained by coating them with a mixture of ZDS and FLC. We
followed again a two-step ligand exchange procedure. Initially, as-synthesized OlAc- NPs (0.804
mg) were treated with a solution of FCL (5 mg) in methanol (1 ml) and stirred at 45 °C for 6 days.
Then, a solution of ZDS (5 mg) in DMF/water 8:5 (1.3 ml) was added to the NPs and the mixture
53
was mixed at 70 °C for 16 h. The NPs were precipitated with acetone, dispersed in DI water and
afterwards purified by dialysis for 48 h against DI water (MWCO 12500 Da).
h.07. Evaluation of NP cytotoxicity
The assays to evaluate the NP cytotoxicity were performed in the log phase of growth after the
HepG2 cells had been placed in 24-well plates (35,000 cells/cm2) where they stayed for 24 hours.
Aqueous solution of iron oxide NPs were added to the cell medium to final concentrations of 2.5, 5,
10, 25, 50, and 100 µg Fe/ml and left for 24 h. All experiments were performed in quintuplicate.
h.08. Evaluation of NP uptake by HepG2 cells
h.08.1 Qualitative determination of NP internalization
To carry out qualitative determination of NP internalization by Prussian Blue staining, HepG2 cells
were placed on 24 mm glass coverslips (52,000/cm2) for 24 h before the treatment with NPs, which
were added at the desired concentration to a final volume of 2 ml of culture medium. The day after,
all non-internalized NPs were washed away with large amount of PBS buffer and fixed for 30 min
in a solution of paraformaldehyde (4% PFA). Then, coverslips were exposed for 30 min to freshly
prepared Perls’ reagent (4% potassium ferrocyanide / 12% HCl, 1:1 v/v), washed with PBS and
then held onto slides for examination under an optical microscope (Axioplan, Zeiss) coupled to a
CCD camera (AxioCam, Zeiss). Bright-field pictures were acquired at 63x magnification.
h.08.2 Quantitative iron evaluation
To perform quantitative determination of NP internalization, HepG2 cells were placed on 10 cm-
plates, 45,000/cm2 for 24 hours and then treated for 24 h with NPs at a final concentration, in the
culture medium, of 0, 2.5, 10, 25, 50 or 100 µg Fe/ml. Afterwards, HepG2 cells were washed five
times with PBS to remove the non-internalized NPs and separated from plates through trypsin
treatment. Pellets were recovered after 5 minutes centrifugation at 800 g and suspended in DI water.
Their protein content were quantified by the BIORAD-Dc protein assay. To carry out the
quantitative determination of iron in the pellets, the cell pellets were digested with a mixture of
54
conc. HCl / conc. HNO3 3:1 v/v able to oxidize the organic matter and transform the iron oxide NPs
into soluble Fe3+
ions. We evaluated by UV-Vis spectroscopy37
the formation of the stable red
complex Fe(tiron)3 (tiron = disodium 4,5-dihydroxy-1,3-benzenedisulfonate) at pH 7. We
analyzed its absorbance considering the UV-Vis spectral profile between 400 and 800 nm39
.
In more detail, the cell pellet was dehydrated, treated with high-purity concentrated acids (150 l
HNO3 and 450 l HCl), heated in a gradual manner to boiling, added with 300 l of HCl and again
evaported to dryness. We repeated this procedure 3 times. The solid residue was left at RT during
the night with acids (150 l HNO3 and 450 l HCl). The day after, the digested residue was again
heated to boiling, added with 300 l of HCl and dried. Finally, the residue was recovered with 1 ml
of 0.1 M HCl and dissolved in 10 ml of milliQ water. A sample (3 ml) of the aqueous solution was
treated with powdered PBS until pH 7 and then treated with excess tiron (2 mg). One hour after
the tiron addition, we recorded the UV-Vis spectrum of the sample. We had previously calibrate the
procedure by measuring a series of diluted Fe3+
standard solutions at different concentrations in PBS
buffer. In this way we had obtained the molar extinction coefficient ε(λ) of the Fe(tiron)3 complex
in the 400-800 nm range. The Levenberg-Marquard least-squares method was employed to fit the
spectrum of the unknown sample to the ε(λ) curve.
h.09. Intracellular iron localization
Cells treated with fluorescent NPs were fixed for 30 min in a solution of paraformaldehyde (4%
PFA) and then stained with DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride). Finally the
coverslips were placed onto microscope slides for examination under a confocal microscope (LSM
510 Meta, Zeiss) at 63x magnification. Instead for Lysotracker labeling, the cell medium of HepG2
exposed for 6 or 24 hours to NP dispersions was removed and replaced with fresh medium
containing 1 μM Lysotracker-Red (Molecular Probes). Then, cells were incubated for 30 minutes in
the dark at 37 °C and washed with PBS before staining.
55
i. Characterization methods and instruments
1H- and
13C-NMR spectra were registered with a Bruker Avance spectrometer in (
1H: 400 MHz,
13C: 100 MHz). Transmission electron microscopy (TEM) images were taken with a Zeiss LIBRA
200FE microscope (Carl Zeiss AG, Oberkochen, Germany). The TEM sample was obtained by
evaporating in air a drop of diluted NP solution on a carbon covered copper grid. We measured the
diameter distribution of the nanocrystal core using the software PEBBLES from TEM images
31.UV-Visible spectra were recorded on a Thermo Scientific Evolution 600 spectrophotometer.
FTIR spectra were registered with a Thermo Nicolet NEXUS 670 FTIR spectrometer (Thermo
Fisher Scientific, Waltham, MA, USA). The sample for FTIR was obtained by grinding and
pelleting dry NPs with KBr (NP:KBr 1:100 w/w) or by evaporating a drop of NP solution on a thin
silicon substrate. TGA curves were recorded employing a Perkin-Elmer 7HT instrument (Perkin
Elmer Waltham, MA, USA). In particular the weight loss was monitored while heating up the
sample in air from 50 °C to 900 or 1000 °C at a rate of 5°C/min. Hydrodynamic diameter and ζ
potential of NPs were measured at 25 °C using a Zetasizer Nano ZS (Malvern Instruments Corp.,
Malvern, Worcestershire, UK) at a wavelength of 633 nm (solid state He-Ne laser) and at a
scattering angle θ = 173°; instead scattering intensity Is was measured employing a BI 90 Plus
(Brookhaven Instruments Corporation, Holtsville, NY, USA) at θ = 90°. Moreover the NP
dispersion had a final concentration of 50 µg Fe/ml medium, both in deionized water and in RPMI
(serum-free and complete), since it was an usual value employed in the HepG2 internalization tests.
We monitored the conditions at the start and at the end of the NP uptake experiments measuring
DLS respectively after NP addition and after 24 h incubation in medium. A gentle shaking before
the DLS analysis was made to ensure the sample homogenization and we took at least three
measurements for each sample.
56
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60
o Part II: Magnetic NPs coated with functionalized
polyethylene glycol (PEG)
01. Introduction
Until now, the protection and/or modification of the NC surface with polymers such as
polysaccharides, polyacrilamide, poly(vinyl alcohol) and poly(ethylene glycol) (PEG) is the most
common method used to ensure to the NPs the appropriate pharmacokinetic features for a specific
application in vivo (such as good colloidal stability, reduced biomolecular corona formation, and
increase of the NP half life)1.
In particular, NP functionalization with biocompatible PEG chains, a method known as
“PEGylation”, is currently applied to improve the “stealth” properties of nanosystems which are
known as the “enhanced permeation and retention” (EPR) effect of PEGylated surfaces.
Chemically, PEG is a polymer of the ethylene glycol monomer (HO-CH2-CH2-OH). It is
commercially available in a range of different molecular weights from several hundreds to a few
thousands of Dalton.
PEG has some well-known favorable features: (i) excellent solvating properties, (ii) good stability
against oxidation, reduction, and decomposition and (iii) the possibility to modify it with different
terminal groups useful to attach large biomolecules such as antibodies, drugs, fluorescent TAGs (
for example by the selectively oxidation of the PEG terminal OH moiety1). Moreover, a PEGylated
coating shields the surface charge of nanosystems, enhances water solubility and provides
flexibility to the NPs. These features, together with PEG high surface density, determine the ability
of PEGylated NPs to avoid non-specific opsonin absorption and the subsequent internalization by
the reticuloendothelial system (RES), also known as mononuclear phagocyte system (MPS). PEG
chains, both covalently and electrostatically bound to the NP surface, can show effective protein
rejection tendency2. Nevertheless, the higher stability of covalent bonding can ensure that the PEG
coating maintain its properties in vivo, during blood circulation, or when the NPs are stored in ionic
media. The use of PEG as surfactant increased also thanks to its non-immunogenicity and the
knowledge of full toxicity profiles of some PEG compounds. In the literature the most appropriate
molecular weight for PEG to achieve the EPR effect has been reported to be in the 1500–5000 Da
range3. However, such large molecular weight increases considerably the NP hydrodynamic
diameter4, thus being a limitation in the use of PEG since even NPs based on very small core
nanocrystals could not be excreted by kidneys and would accumulate in the body. In addition, long
61
PEG chains may also entrap biomolecules potentially present on the NP surface and interfere with
their presentation to the environment.
As depicted in Fig 1, NPs can be PEGylated by 1) direct PEGylation ( PEG molecules are adsorbed
at the NP surface via physical bonding during the synthesis of NPs using PEG as solvent or other
thermal/hydrothermal methods), 2) covalent attachment by anchoring groups. The latter strategy
includes (i) monofunctional PEG (this way is very effective for inorganic materials characterized by
high and selective binding affinity towards a specific group, such as gold NPs for thiol –SH
groups), and (ii) bifunctional PEG molecules (which, besides a grafting group, have a moiety useful
to achieve functionalization of the NP with selected ligands for theranostic features)1. Inorganic
NPs such as metal and metal oxide NPs are usually PEGylated in these ways.
Fig 1. Various strategies for the PEGylation of NPs. All the strategies result in NPs that are water
soluble and can repel opsonin proteins. Direct PEGylation (by physical or electrostatic adsorption)
has the advantage of a simple synthesis. Monofunctional PEGs can be used to achieve covalent
bonding between the PEG molecules and the NPs providing long-term stability and high dispersion
stability. The vectorization of NPs can be achieved by using bifunctional PEG molecules wherein
the free terminal functional groups of PEG can be covalently grafted to other polymers, fluorescent
tags, and targeting antibodies or proteins. Image from Ref 1.
62
As mentioned before, thiol (–SH) terminated PEGs are used to coat gold NPs because of the very
strong binding affinity between Au and SH moieties (S-Au bond energy = 47 kcal mol-1
). PEG
molecules with molecular weight below 5000 Daltons are often preferred over higher molecular
weights. Another important parameter is the spacer length of the PEG, for example when
fluorescent tags are also present in the NP coating, because of the spacer length-related change of
fluorescence intensity. As Ti and coauthors reported, PEG5000 chains could be covalently linked to
Au NPs through thioctic acid (TA), a compound containing a cyclic disulfide5. The presence of TA
increased the NP stability in phosphate buffer saline, stability which was also controlled by NP size
(20 and 40 nm Au NPs showed a lower tendency toward aggregation than 80 nm NPs and a delayed
clearance from the blood). These results were ascribed to a lowest surface PEG density of 80 nm Au
NPs. Nanosystem instability might come from oxidation of thiolated species and from exchange
reactions with other thiol compounds present inside the body.
Fig 2. (A) Representative pancreatic cancer tissue thin sections labeled using secondary antibody
staining (red regions). (B) Dark field transmission scattering images of GNP-F19-labeled
pancreatic cancer tissue, (C) GNP-F19-labeled healthy tissue, and (D) GNP-mIgG-labeled
pancreatic cancer tissue. The expanded section in B has been contrast enhanced to emphasize the
presence of individual GNP-F19.Image from Ref 6.
63
Homo and hetero bifunctional PEG can be employed to covalently bind a specific functionality. For
example Mason et al6 synthesized ≈ 15 nm spherical Au NCs coated with PEG ligands having a
dithiol group for grafting to the NC surface and a terminal carboxy moiety for binding F19
monoclonal antibodies. The NP dispersions did not aggregate for a long time as demonstrated by
DLS, size exclusion chromatography, and TEM analysis. Finally the authors demonstrated the NP
ability to selectively stain a human pancreatic cancerous tissue (Fig 2).
Focusing now on magnetic iron oxide NPs, those produced by aqueous routes can be directly
PEGylated. For example, Reimhult et al. stabilized NPs obtained by aqueous precipitation using
methoxy-PEG(550)-gallol, methoxy-PEG(5000)-6-hydroxy-dopamine, biotin-PEG(3400)-6-
hydroxy-dopamine or their mixtures7 (Fig 3). The NPs can be freeze dried thanks to the high
binding affinity of the trihydroxy-benzene groups toward iron oxide surface, stored for at least 20
months and easily redispersed in water as individual particles when necessary. To produce MRI
contrast agents, the authors functionalized their NPs with anti-human vascular cell adhesion
molecule 1 (VCAM-1) antibodies binding them to the biotinylated PEG-gallol units through
neutravidin. VCAM-1 was chosen since it is over expressed at the endothelial cell periphery of
atherosclerotic sites, aging as early marker of this disease.
Fig 3. Schematic image of PEG-gallol stabilized iron oxide nanoparticles where m ≈10 and n ≈73.
Approximately 9 mol% of the adsorbed PEG-gallol was biotinylated. Image from Ref 7.
In another article Reimhult and coauthors have extensively analyzed the capacity of eight different
PEG5000-based mono-catechols to act as anchors for iron oxide NPs8. Stability tests in 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.2) containing 150 mM NaCl
64
showed that electron-attractive groups, such as nitro-group, present on the catechol ring, were
effective in enhancing the NP stabilization.
Going back to the NP PEGylation strategies, the direct PEGylation, in addition to small and
uniform NC size, was obtained in the literature also by a non-hydrolytical synthesis. In particular,
Fe(acac)3 was thermally decomposed in 2-pyrrolidone and in the presence of monocarboxyl-
terminated poly(ethylene glycol) (MPEG-COOH)9, which results as covalently bound to the NP
surface by COOH moieties. NPs displayed excellent water solubility and biocompatibility and so
they were tested as magnetic resonance imaging (MRI) contrast agents.
Instead, NPs produced by non-hydrolytically routes are typically coated with hydrophobic
molecules such as oleic acid or oleylamine10
. Therefore, in order to PEGylate these systems it is
necessary to follow different exchange procedures in which the new surfactant is typically formed
by two parts, a region grafting the NP surface and the PEG chain as hydrophilic part exposed to the
aqueous medium (named as “bifunctional ligand” in Fig 4).
Fig 4. Surface modification strategies for designing MNP probes with high colloidal stability.
Image from Ref 4.
For example Sun et al11
synthesized monodisperse Fe NPs through thermal decomposition of
Fe(CO)5 in the presence of oleylamine in octadecene at 180°C. Then the NP dispersion in hexane
65
was exposed to air in order to oxidize the iron surface producing a crystalline Fe3O4 shell. The
oleylamine exchange with PEG-dopamine leads to a stable aqueous NP dispersion in PBS, useful
for biomedical application (Fig 5).
Fig 5. c) Exchange of ligands on Fe@Fe3O4 nanoparticles for PEG–dopamine. d) TEM image of
water-soluble Fe@Fe3O4 nanoparticles. Images from Ref 11.
The colloidal stability of NP solution was further increased thanks to employment of multidentate
polymeric ligands, since the approach of increasing the number of grafting groups in a NP
surfactant allows to minimize its loss. For instance Matoussi et al12
have developed some oligomers
named as OligoPEG-Dopa, since they were formed by several catechol groups binding NC surface,
a short poly(acrylic acid) backbone and polyethylene glycol chains. In addition, PEG allowed
further coupling with different biomolecules, for example by azide-alkyne cycloaddition when the
PEG had an azide end-group. These oligomers gave quick exchange process and exhibited a better
colloidal stability varying the pH range and in the presence of electrolytes, compared to other
olygomers having several carboxyl groups or to monodentate ligands having a catechol or a
carboxyl moiety. The authors verified also the ability of magnetite NPs coated with OligoPEG-
Dopa to act as T2 contrast agents and tested their cytotoxicity in live cells which resulted low
significant. Bawendi and coauthors showed another example of multidentate ligand, a PEG-
polymeric phosphine oxide ligand which was used to transfer various NPs (Au, γ-Fe2O3, Pd, and
QD) from organic solvents to water maintaining their physical properties and reactivities13
.
Considering the well-known ability of the catechol group and its derivatives to bind with high
affinity iron ions and give stable NP water dispersion14-15
, in this PhD thesis we have decided to
66
choose this grafting group for our NP dispersion. Moreover about PEG chain molecular weight, we
resorted to PEG 5000.
We would also functionalize magnetic NPs with the Fab fragment of Trastuzumab (Herceptin®),
which is a monoclonal antibody specifically binding the human epidermal growth factor receptor 2
(HER2). Such receptor is up-regulated in 20-30% of breast cancer cells and also in some classes of
adenocarcinoma cells of the gastro-esophageal junction and of stomach. In this case the large
biomolecule provides the NPs with (i) an active targeting since the antibody strongly binds to its
antigen and (ii) therapeutic features since Trastuzumab affects the cellular proliferation mechanism
in different ways. In a future application in cancer therapy, the functionalization of the iron NPs
with Fab Trastuzumab will improve treatment specificity and reduce toxicity and side effects
typical of a traditional chemotherapy. The Fab fragment was produced by Bioker srl; it has a
molecular weight around 5.6 kDa and a dimension of 5 nm. Therefore we have designed and tested
different catechol molecules: (i) a bi-functional PEG displaying besides to the catechol anchor
moiety, a maleimide terminal group in order to react with the free thiol group of the Fab
biomolecule; (ii) mono and bi-dentate polymeric ligands in order to employ them as co-surfactants
to dilute the bi-functional linker described before at the NP surface. Their chemical structures will
be presented in detail in the next Chapter, together with the description of the procedures used to
obtain PEGylated NPs and their characterization.
67
02. Preparation of iron oxide NPs coated with functionalized
polyethylene glycol
a. Synthesis of magnetic nanoparticles
Two synthetic strategies were followed in order to prepare magnetic iron oxide NPs, hot-injection
of metal precursor and co-precipitation of iron salts.
a. 01. Synthesis of magnetic NPs by hot-injection
We prepared monodisperse spherical iron oxide NPs coated with oleic acid (diameter 9.6 nm)
following a hot-injection method in 1-octadecene (ODE) described in the literature (see
Experimental Section) with a molar ratio between metal precursor and oleic acid equal to 1:316
.
Fig 6. OlAc-coated NPs synthesized by hot-injection. Left) TEM image. Right) Diameter histogram
(as measured by the software PEBBLES17
). The median diameter is 9.6 nm.
As shown in Fig 6, the median diameter measured from the TEM images was <d> = 9.6 nm with a
diameter standard deviation σd = 0.9 nm, corresponding to a dispersion σd/<d> = 8.5 %.
These iron oxide NPs had a spinel structure (Fig 7) and stoichiometry intermediate between
magnetite (Fe3O4) and maghemite (Fe2O3).
4 5 6 7 8 9 10 11 12 13 14 15 160
200
400
600
800
1000
1200
Equiv. Diameter (nm)
NP
counts
Equiv. Diameter (nm)
mean: 9.7
median: 9.6
st.dev.: 0.8 (8.5%)
No. of NPs: 1811
68
Fig 7 . Electron diffraction pattern of OlAc-coated NPs synthesized by hot-injection.
a. 02. Synthesis of magnetic NPs by co-precipitation
Iron oxide NPs are typically obtained on a large scale by co-precipitation from a stoichiometric
mixture of Fe3+
and Fe2+
salts in alkaline aqueous media. This method for the synthesis of magnetite
NPs by precipitation of FeCl3 and FeCl2 with alkali was introduced by Massart18
. The possibility of
a large-scale preparation along with the use of “green chemistry” methods, that avoid the
employment of toxic reagents, are appealing prerequisites for a scalable synthesis of magnetic NPs
to industrial applications for medical use19
. In this case, the resulting NPs are free from surfactants,
so in principle more suitable to being directly dispersed in water after coating by hydrophilic
molecules such as PEG ligands. Precursor, reaction temperature, and pH are the experimental
parameters to control NP morphology and dimension but it is however difficult to synthesize NPs
with a narrow size distribution20
. Other drawbacks of these magnetite NPs are their instability and
poor crystallinity, with a loss in magnetic susceptibility19
. We used as metal precursor FeCl3.6H2O
and FeSO4.7H2O. The followed synthetic procedure was described in detail in the Experimental
Section. As clearly visualized in TEM image (Fig 8), our NPs are polydisperse according with those
produced in the literature. The measured NP size was 2.9 nm with a diameter standard deviation σd
= 0.8 nm.
69
Fig 8. TEM image (left) and electron diffraction pattern (right) of “naked” iron oxide NPs obtained
by co-precipitation.
70
b. Synthesis of catecholic PEG ligands
We have designed the organic preparation of different types of PEG 5000 catecholic ligands whose
structures are shown below (Fig 9). In our strategy, we attributed to them different roles as
surfactants: in particular, structure (a) refers to the bi-functional linker which we are going to bind
to the Fab fragment of Trastuzumab thanks to its maleimide end group; structures (b) and (c)
display possible co-surfactants of the bi-functional linker. In this case, we want to employ ligands
chemically similar to the bi-functional linker and in addition, with compounds (c), to explore the
effect, if present, of a bidentate grafting group on the NP colloidal stability. Indeed, we could
potentially improve the NP water stability by increasing the number of grafting groups per ligand
and so decreasing the chance that a surfactant molecule is irreversibly lost from the NP coating.
HO
HO
O
OO
n
HO
HO
ONH
O
O
PEG5000 NH
N
O
O
O
NHNHO
HOO PEG5000OMeR
R = H
OH
OHH2CR =
Fig 9. Chemical structures of the desired PEG5000 catecholic ligands displaying different functions:
the bi-functional linker (a) to bind biomolecules; co-surfactants (b) and (c).
b. 01. Synthesis of catechol (a) and (b) compounds
The synthesis of compounds (a) and (b), named as SS33 and SS20_A respectively, were made in
collaboration with the laboratory of CISI scrl inside the RSPPTECH project (“Ricerca e sviluppo di
(a)
(b)
(c)
71
prodotti e piattaforme tecnologiche per la competitività dell’industria Lombardia) of Regione
Lombardia that financially supported us (see the detail in the Experimental Section).
b. 02. Synthesis of catechol (c) compounds
We designed the linker structures of (c) starting from the ethylenediamine scaffold. One of the
nitrogen atoms of ethylenediamine is linked to the PEG 5000 chain while the other nitrogen atom is
bound to one or two (3,4-dihydroxyphenyl)methyl moieties. We could bind the PEG chain to the
central amine scaffold (I) before the introduction of the 3,4-dihydroxyphenyl groups or (II) as the
final reaction step, by coupling it to mono- or di-catechol-diamine fragment. In both these
approaches, we planned that the PEG chain is going to react with one of the nitrogen atoms of
ethylenediamine as the brominated PEG derivative 12 (see Scheme 3 in the Experimental Section).
We obtained the compound 12 following the procedure reported in literature by Cozzi et al21
in
order to achieve, from the starting material 9, whose hydroxyl group is not reactive enough because
of steric hindrance, substrate 11 that is more reactive in SN2 reactions, thanks to the introduction of
the 3-(4-hydroxyphenyl)propanol spacer. Considering that in the general type-(I) strategy, the
isolation and characterization of the intermediate substances could be more difficult due to the high
molecular weight of the PEG chain, we decided to follow the type-(II) procedure visualized in
Scheme 4 in the Experimental Section in order to produce the desired mono-catechol 16.
Since many protecting groups for the 3,4-dihydroxyphenyl moiety were in principle appropriate, we
have synthesized different examples of hydroxyl-protected derivative 13, first exploring the R =
tert-butyldimethylsilyl (TBDMS) ether. The derivative 13_TBDMS was synthesized starting from
3,4-dihydroxybenzaldehyde 17 following the Scheme 5 depicted in the Experimental Section.
However, when compound 14_TBDMS was treated with 12, according to the general synthetic
pathway (Scheme 4) proposed to achieve the mono-catechol surfactant, the reaction did not proceed
and we did not obtain 16. Therefore, we tried to investigate in detail this step to clarify if the reason
was again a poor reactivity of a high molecular weight PEG derivative. With this purpose we
designed the reaction reported in the Scheme 6 of the Experimental Section, where the compound
14_TBDMS was treated with compound 21 directly derived from the 3-(4-hydroxyphenyl)propanol
spacer. Also in this case, although in absence of the long PEG chain, 1H-NMR and HPLC-mass
analysis confirmed that the reaction did not proceed to the desired product 22 and gave a mixture of
byproducts.
For this reason, we modified the chosen protecting group R focusing on the acetonide functionality.
The protected derivative 13_Acetonide was prepared from 3,4-dihydroxybenzoic acid 23 (see
72
Scheme 7 in the Experimental Section). Unfortunately, we could not proceed beyond molecule 14-
Acetonide because it did not react with 12 as also occurred with the derivative 14_TBDMS. We
have correlated these negative results to the - at least partial - deprotection of both tert-
butyldimethylsilyl ether and acetonide protecting groups in our reaction conditions. So, we finally
resorted to the benzyl ether protecting group, which we have already employed in our previous
work22
to obtain a PEG-based tetra-catechol surfactant, hoping that it can be more stable in our
reaction conditions. As shown in the Scheme 7 of the Experimental Section, with this protecting
group, in addition to 13_BnO bromide, we want also to consider the 13_BnO chloride and
13_BnO mesylate, trusting that their dissimilar SN2 reactivities could allow us to achieve, in a
controlled way, both the mono- or bi- cathecol linkers (c) by nucleophilic substitution on the
ethylendiamine scaffold. These experiments are in progress.
73
c. Preparation of water-soluble PEG-ylated NPs
To prepare PEG-ylated NPs, we employed the previous batches of magnetic iron oxide NPs
synthesized by hot-injection and co-precipitation.
In the first case, we followed a ligand exchange procedure in order to displace the pristine oleic acid
with the desired PEG molecules whereas in the second case the “naked” NPs were directly treated
with PEG surfactants (see Experimental part). Here I reported TEM image ( Fig. 10) of iron oxide
NPs produced by co-precipitation and coated with a mixture of bi-functional linker (a) and co-
surfactant (b). The measured NP size was 2.6 nm with a diameter standard deviation σd = 0.7 nm.
Fig 10. TEM image (left) and electron diffraction pattern (right) of iron oxide NPs produced by co-
precipitation and coated with a mixture of bi-functional linker (a) and co-surfactant (b).
74
d. Characterization of PEG-ylated NPs
We have characterized PEG-ylated NPs by different techniques. First, we carried out Fourier-
Transform Infrared (FTIR) spectra of PEG molecules SS33 and SS20_A alone. As expected, the
FTIR spectra are similar spectra since they are dominated by the presence of the high molecular
weight PEG chain (Fig 11).
Fig 11. Comparison between IR Spectra of SS33 and SS20_A PEG molecules.
In particular, we could observe the vibrations of the PEG which comprise CH2 bending (1467
cm−1
), CH2 wagging (1344 cm−1
), CH2 twisting (1281, 1251 cm−1
), C–O and C–C stretching (1116
cm−1
), and CH2 rocking (953.6, 841.8 cm−1
)22
. In the spectrum of PEG molecule SS33 we can
appreciate also the presence of a strong band of at 1705 cm−1
arising from the out-of-phase
stretching of C=O of maleimide moiety.
In addition IR spectra allow us to confirm that the exchange process was effective since iron oxide
NPs appeared coated with PEG derivative (Fig 12, 13, 14), without bands coming from the pristine
oleic acid surfactant.
75
Fig 12. Comparison of IR spectra of PEG derivative SS20_A and iron oxide NPs coated with it,
after the exchange process.
Fig 13. Comparison of IR spectra of PEG derivative SS33 and iron oxide NPs coated with it, after
the exchange process.
76
Fig 14. Comparison of IR spectra of PEG derivative SS20_A, PEG SS33 and iron oxide NPs coated
with a mixture of them, after the exchange process.
We have also measured by DLS the (intensity weighted) hydrodinamic diameter of the PEGylated
NPs, after dialysis and filtration, that resulted around 240 nm for co-precipitated NPs and 120 nm
for solvothermal NPs (see Experimental Section).
By measuring the iron content of our NP solution with UV-Vis method described in the Part I, we
have evaluated that the yield in water soluble NPs is approximately the same, around 50%.
Instead by calculation of the percentage of organic portion present at the NP surface from CHN
analysis data, we deduced that this value is higher for NPs obtained by co-precipitation (around
90%) than that of solvothermal NPs (around 70%) (See Experimental Section).
Finally, having the idea of functionalizing the nanosystems with the Fab fragment of Trastuzumab,
we need to investigate and potentially quantify the reactivity of maleimide groups of the bi-
functional linker (compound (a) or PEGSS 33) present in the NP coating.
In particular, we carried out such quantification with a commercially available fluorescent kit
(AmpliteTM
fluorimetric maleimide quantitation kit) able to measure protein maleimide groups.
As the producers specified, this kit employs a dye that has enhanced fluorescence after reaction
with a maleimide. It is able to detect as little as 100 nM in concentration of maleimide, performing
the analysis directly in a 96-well-plate and choosing Ex/Em = 490 nm/520 nm. By comparison with
77
a shorter maleimido PEG (molecular weight ≈ 2000 Da), we have concluded that maleimide moiety
was less reactive in the longer PEG5000 chain. However, a fraction of maleimide group exposed at
the NP surface was actually reactive and sufficient to functionalize the NCs with the desired amount
of anti-body fragment.
78
03. Conclusions
In the second part of my PhD thesis, we have explored examples of PEGylated molecules in order
to ensure colloidal stability to our nanosystems, as indicated in the literature.
In particular, we have also designed a compound, the bi-functional linker, potentially able to react
with large biomolecule, such the Fab fragment of the monoclonal antibody Trastuzumab.
In this way we are trying to achieve iron oxide NPs provided with an active targeting.
From a synthetic point of view our efforts are focused on producing water-soluble PEG-ylated
linkers with one or two catechol grafting group(s) that we want to use as co-surfactants for the bi-
functional linker, having the same chemical nature and preserving the good water solubility and
stability of our nanosystems. Although several protecting groups for the catechol moiety are in
principle usable, we have proved that in our experimental conditions, some of them (tert-
butyldimethylsilyl ether and acetonide) are at least partially deprotected. Therefore, we have
selected the benzyl group as a promising protecting group for catechol moieties.
In addition to these synthetic studies, we have produced and characterized two batches of iron oxide
NPs coated with a mixture of the bi-functional linker and a catechol-PEG5000-OMe acting as co-
surfactant. One batch of NPs was produced by hot-injection of Fe(CO)5 in 1-octadecene and in the
presence of oleic acid, while the other by co-precipitation of iron salts in water. Solvothermal NPs
showed a good size dispersion and crystallinity, whereas co-precipitated NPs can be produced in
large amount and in “green chemistry” conditions. In both cases, the yield in “water soluble NPs”
was estimated around 50%. Indeed, evaluating by CHN analysis the amount of PEG molecules
present in their coating, this was higher for NPs produced by co-precipitation.
Finally, thanks to a commercially fluorescent kit, we have deduced that the maleimide moiety was
less available for reaction in the longer PEG chain than in the shorter one (molecular weight around
2000 Da). However, a fraction of the maleimide groups present at the NP surface was reactive,
enough to functionalize the NCs with the desired antibody fragment avoiding problem arising from
steric hindrance.
Therefore, as already deduced for zwitterionic iron oxide NPs, also PEGylated NPs could be
considered promising in the development of theranostic nanosystems.
79
04. Experimental section
e. Materials
All chemical and solvents were acquired from Sigma-Aldrich and used without further purification.
Aldrich Trace Select concentrated acids (HNO3 and HCl) were employed to digest NPs.
f. Procedures
f.01. Acronyms used in the experimental section
Fe(CO)5 = iron pentacarbonyl
RT = room temperature
NaOH = sodium hydroxide
FeCl3.6H2O = iron (III) chloride hexahydrate
FeSO4.7H2O = iron (II) sulfate heptahydrate
HCl = hydrochloric acid
PEG 5000 = polyethylene glycol with molecular weight around 5000 D
PPTS = pyridinium p-toluenesulfonate
EtOAc / AcOEt = ethyl acetate
LiAlH4 = lithium aluminium hydride
THF = tetrahydrofuran
Na2SO4 = sodium sulfate
CH3OH/ MeOH = methanol
NH3 = ammonia solution
DCM = dichloromethane
CD3OD/ MeOD = deuterated methanol
CDCl3 = deuterochloroform
K2CO3 = potassium carbonate
PBr3 = phosphorous tribromide
H2O = water
CH3CN = acetonitrile
TEA = triethylamine
TBDMS-chloride = tert-butyldimethylsilyl chloride
NaBH4 = sodium borohydride
80
Et2O = diethyl ether
HBr = hydrogen bromide
H2SO4 = sulfuric acid
EtOH = ethanol
NaHCO3 = sodium hydrogen carbonate
DMP = 2,2-dimethoxypropane
SOCl2 = thionyl chloride
CHCl3 = chloroform
f. 02. Synthesis of magnetic NPs by hot-injection
We obtained 9.6 nm iron oxide NPs according to a published literature procedure16
. In particular
Fe(CO)5 (12 ml) was injected in a mixture of 1-octadecene (45.96 g) and oleic acid ( 7.68 g, molar
ratio metal precursor: surfactant 1: 3) at 120°C which was then heated up to 320°C for 3h under
nitrogen. After cooling at RT, the NPs were collected by precipitation with acetone and
centrifugation, repeatedly washed with acetone and finally solved in toluene.
f. 03. Synthesis of magnetic NPs by co-precipitation
We prepared iron oxide NPs following a method reported in the literature23
using sodium hydroxide
instead of the ammonium base to co-precipitate ferrous and ferric ion water solutions in
stoichiometric molar ratio (1:2). 4.17 ml of NaOH 10M was injected inside a solution of
FeCl3.6H2O (0.65 g , 2.4 mmol) and FeSO4.7H2O (0.333 g, 1.2 mmol) in water (25 ml) and
concentrated HCl (130 l) under mechanical stirring. Immediately a black precipitate formed and
was stirred for 1 hour. Then iron oxide NPs were collected by centrifugation, washed with
deionized water until the pH became neutral and dispersed in 26 ml of water (concentration ≈ 10
mg of Fe3O4 /ml water as used by Reimulth and coauthors in following exchange process with
mono-functional catechol PEG 50008).
f. 04. Synthesis of the bi-functional linker (SS33)
The bi-functional linker (SS33) was produced inside the laboratory of CISI scrl following the
Scheme 1 reported below and starting from the product commercially acquirable (1).
81
Br N
O
OHOHO OH
OH
O O
O
OON
O
OOH2N
N (CH2)5
O
O
HN
O
PEGO
O
O
N (CH2)5
O
O
HN
O
PEGO
O
NH
O
O
O
N (CH2)5
O
O
HN
O
PEGO
O
NH
O
OH
OH
N
O
O
PPTS
Toluene dry170 min reflux, on rt
, ,Cs2CO3
DMF dry2 h, rt
LiAlH4 in THF 1M
THF dry30 min, rt
DCM dry3 h, rt
water,2 h, refluxHCl 6N
1 2 3 4
5
6
Scheme 1. Synthetic pathway leading to the SS33 (6).
f. 04. 1 Synthesis of compound 2
The product 2 was produced according with a method reported in literature24
and employing a two-
necked flask, furnished with a distillation apparatus in one neck and a stopcock in the other. A
mixture of benzene-1,2,4- triol (0.4g, 3.17 mmol), pyridinium p-toluenesulfonate (0.64 mg,
2.54x10-3
mmol), and dry toluene (32 mL) was stirred and heated to reflux with distillation of the
solvent. During the reaction 2,2-dimethoxypropane in portion (0.096 mL x 4; 0.064 mL x 4; 4.63
mmol) and dry toluene (to compensate for the loss of solvent) were added to the mixture every 15
min. After 3h, the reaction mixture was cooled to r.t. and stirred over night. The crude was
subjected to column chromatography (silica gel, petroleum ether 100%→ petroleum ether /EtOAc
9.5/0.5) to give 2 (0.296 g, yield 56 %) as a light yellow oil.
82
f. 04. 2 Synthesis of compound 3
Bromoacetonitrile (0.101 ml, 1.46 mmol) was added to a dispersion of compound 2 (0.121 mg, 0.73
mmol) and Cs2CO3 (0.476g, 1.46 mmol) in dry DMF (5 mL), cooled at 0°C. Then the mixture was
stirred at r.t. for 2h, filtered through celite and DMF was removed under vacuum. The crude was
purified by flash chromatography (silica gel, isocratic elution with DCM/ EtOAc 1:1). The resulting
product appeared as a yellow oil ( 0.11 g, yield 74%).
f. 04. 3 Synthesis of compound 4
LiAlH4 in THF 1M (1ml, 1 mmol) was added under N2 drop by drop to a solution of compound 3
(0.102 g, 0.5 mmol) in dry THF (5 ml), cooled at 0°C. After that the reaction was mixed at r.t. for
30 min and then stopped by the addition, at 0°C, respectively of water (0.038 ml), 15% NaOH
(0.038 ml) and again water (0.0114 ml), in order to induce the precipitation of lithium salts. Then
the dispersion was stirred at r.t. for 15 min, dried with anhydrous Na2SO4, filtered off and the
solvent was finally removed by evaporation. The crude was purified by chromatography (silica gel,
EtOAc/ MeOH 9/ 1 + 1% NH3), ( 0.05 g, yield 48%).
f. 04. 4 Synthesis of compound 5
A solution of compound 4 (0.044g, 0.21 mmol) in anydrous DCM (2 mL) was additioned dropwise
to a solution of Malhex-NH-PEG-O-C3H6-CONHS (0.891g, 0.18 mmol) in dry DCM (2 mL)
refrigerated at 0°C. The mixture was stirred at r.t. for 3h. Then the product was precipitated by
adding diethyl ether ( 84 ml), stirred at r.t for 1h, collected by filtration and washed with fresh
diethyl ether. In order to remove the fraction of Malhex-NH-PEG-O-C3H6-CONHS that did not
react, the crude was solved in DCM and treated with PS-NMM (Biotage®, 0.2g, 0.36mmol)e PS-
NH2 (Biotage®, 0.24g, 0.36mmol).(0.458 g, yield 68%).
f. 04. 5 Synthesis of compound 6
A water solution (10 ml) of compound 5 (0.445 mg, 0.09 mmol) was treated with HCl 6N (3 l),
heated to reflux for 2h under magnetic stirring. Subsequently the reaction was cooled at r.t, and
water was removed by evaporation affording the desired product SS33 (6) (0.421 g, yield 95%). 1H
NMR (400 MHz, MeOD , 25 °C): = 8.1 (m, 1H, CONH-Ar), 7.95 (m, 1H, maleimide-CONH),
83
6.84 (s, 2H, maleimide H ) 6.68 (d, 1H, aromatic ring H), 6.45 (d, 1H, aromatic ring H), 6.30 (dd,
1H, aromatic ring H), 3.97- 3.94 (m, 2H, Ar-OCH2), 3.66 ( PEG chain), 2.35-2.31 (m, 2H,
CH2CONH-Ar ) 2.23-2.19 (m, 2H, maleimide-CH2CONH) 1.90-1.87 (m, 2H, CH2CH2CONH-Ar),
1.69-1.59 (m, 4H, maleimide-CH2CH2CH2CH2CONH), 1.36-1.30 (m, 2H, maleimide-CH2CH2CH2
CH2CONH).
f. 05. Synthesis of the catechol-PEG5000 co-surfactant (SS20_A)
CH3O-PEG-Br
O
OHO
O
OOPEG
H3COCs2CO3
DMF dry,5 h, 90°C MW
OH
OHOPEG
H3COHCl 6N
water4.5 h, reflux
2 7 8
Scheme 2. Synthetic pathway leading to the SS20_A (8).
f. 05. 1 Synthesis of compound 7
CH3O-PEG-Br (0.74g, 0.15 mmol) was added to a mixture of compound 2 (0.027g, 0.16 mmol)
and Cs2CO3 (0.053g, 0.16 mmol) in dry DMF (7 mL). The reaction was heated by a microwave to
90°C. After 5h, the dispersion was filtered through celite and the solvent was evaporated. The crude
was precipitated by treatment with small amount of EtOAc, and then collected by centrifugation;
the purified product appeared as light brown solid (0.65g, yield 83%).
f. 05. 2 Synthesis of compound 8
A water solution (6 ml) of compound 7 (0.648 mg, 0.13 mmol) was treated with HCl 6N (3 l) in
order to remove protecting group acetonide, and heated up at reflux for 5 h. Then water was
evaporated and the residue was crystallized twice with methanol/ diethyl ether leading to the desired
product SS20_A (8) (0.401 g, yield 61%). Additional 0.046 g of product 8 were recovered from the
crystallization water by precipitation with diethyl ether and subsequent filtration thus increasing the
isolated yield to an overall 68%. 1H NMR (400 MHz, MeOD , 25 °C): = 6.68 (d, 1H, aromatic
ring H), 6.45 (d, 1H, aromatic ring H), 6.30 (dd, 1H, aromatic ring H), 4.05- 4.02 (m, 2H, Ar-
OCH2), 3.66 ( PEG chain), 3.38 (s, 3H, OCH3).
84
f. 06. Synthesis of the catechol (c) co-surfactant: synthesis of the
brominated PEG derivative 12
We obtained the compound 12 following the procedure reported in literature by Cozzi et al21
and as
we have already described in our previous work22
. PEG samples were melted at 100°C in vacuum
for 1h before their employment in order to remove traces of moisture. At the end of the reaction, we
roughly purified PEG derivates by evaporating the solvent in vacuum, solving the residue in a small
volume of CH2Cl2 and adding diethyl ether (around 100 mL/g of polymer), under stirring and at
0°C. Then the precipitate was filtered and the solid was repeatedly washed with fresh diethyl ether.
Br
O PEG5000
OCH3
OH
O PEG5000
OCH3
OH PEG5000
OCH3
MsO PEG5000
OCH3
9 10 1112
Scheme 3: Preparation of the brominated PEG derivative 12.
f. 06. 1 Synthesis of compound 10
Mesyl chloride (93 l, 1.2 mmol) dissolved in dry DCM (3 ml) was added dropwise to a solution of
methoxy-PEG5000 commercially available (2g, 0.4 mmol) and trioctylamine (700 l, 1.6 mmol) in
3 ml of dry DCM, cooled to 0°C. The reaction was stirred for 15 min at this temperature, then left
overnight at RT (quantitative yield). 1H NMR (400 MHz, CDCl3, 25 °C) : = 4.40 (m, 2H,
CH2OMs), 3.66 (PEG chain), 3.38 (s, 3H, OCH3), 3.08 (s, 3H, CH3SO3).
f. 06. 2 Synthesis of compound 11
The methoxy-PEG5000 mesylate 10 (10.49 g, 2.07 mmol) was treated with the commercially
available 3-(4-hydroxyphenyl)-1-propanol (629 mg, 4.13 mmol) in anhydrous DMF and in the
presence of K2CO3 (1.43 g, 10.35 mmol).The reaction was heated up to 70°C for 20 h affording the
compound 11 (yield 92%). 1H NMR (400 MHz, MeOD , 25 °C) : = 7.07 (d, 2H, aromatic ring H),
6.82 (d, 2H, aromatic ring H), 4.10 (m, 2H; PEGCH2CH2OAr), 3.66 (PEG chain), 3.57 (m, 2H,
CH2CH2CH2OH), 3.38 (s, 3H, OCH3), 2.63 (t, 2H, ArCH2), 1.82 (m, 2H, CH2CH2CH2).
85
f. 06. 3 Synthesis of compound 12
The compound 11 (1g, 0.195 mmol) was solved in 5 ml of anhydrous DCM in the presence of
triethylamine ( 81 l, 0.584 mmol) and cooled to 0°C with an ice-bath. Then PBr3 ( 55 ml, 0.584
mmol) was added in small portions under magnetic stirring. The reaction was left at r.t. overnight
leading to the desired derivative 12. In this case, in addition to diethyl ether precipitation and
washing, the product was also purified with HPLC using a C18 reverse column. We applied the
following HPLC eluant conditions: from 85% H2O/ 15% CH3CN to 100% CH3CN with a flow rate
15 ml/min (yield 70%).1H NMR (400 MHz, MeOD , 25 °C) : = 7.15 (d, 2H, aromatic ring H),
6.75 (d, 2H, aromatic ring H), 4.10 (m, 2H, PEGCH2CH2OAr), 3.66 (PEG chain), 3.42 (m, 2H,
CH2Br), 3.36 (s, 3H, OCH3), 2.73 (t, 2H, ArCH2), 2.12 (m, 2H; CH2CH2CH2).
f. 06. 4 Synthesis of the mono-catechol (c) co-surfactant (16)
We reported in the Scheme 4 below the synthetic pathway that we wanted to follow in order to
produce the derivative 16. It is an example of type- (II) procedure where the PEG chain is bound to
a mono- or di-catechol-diamine fragment, already obtained.
86
+BrRO
RO
NH2NH2
NHNH2RO
RO
Br
O PEG5000
OCH3
NHNHRO
RO
O PEG5000
OCH3
NHNHOH
OH
O PEG5000
OCH3
13 14
12
15
16
Scheme 4: General preparation of the desired mono-catechol 16.
f. 06. 5 Synthesis of the mono-catechol (c) co-surfactant: tert-
butyldimethylsilyl (TBDMS) ether protecting group
OH
OH
CHO TBDMSO
TBDMSO
CHO TBDMSO
TBDMSO
CH2OH
TBDMSO
TBDMSO
CH2Br TBDMSO
TBDMSO
NHNH2
17 18 19
13_TBDMS 14_TBDMS
Scheme 5: Preparation of the derivative 14 with TBDMS protecting group.
87
f. 06. 6 Synthesis of compound 18
The product 18 was achieved according with a method reported in literature25
starting from the
commercially available 3,4-dihydroxybenzaldehyde 17 (100 mg, 0.72 mmol), solved in dry DCM (4
ml) in the presence of TEA (400 l, 2.88 mmol). The reaction was cooled to 0°C and then a
solution of TBDMS-chloride in a few ml of anydrous DCM was added to the system. After the
addition, we needed to join dry THF to increase the reaction solubility. The mixture was stirred for
4 h at r.t. and evaporated. The residue was treated with water and extracted with diethyl ether. The
organic phase was washed with brine, dried with anydrous Na2SO4, filtered off and finally
evaporated. The crude was subjected to column chromatography (silica gel, hexane/EtOAc 95/5) to
give 18 (0.194 g, yield 74 %).1H-NMR (400 MHz, CDCl3, 25 °C): δ = 9.71 (s, 1H, CHO), 7.28-7.26
(dd, 1H, aromatic ring H), 7.17 (d, 1H, aromatic ring H), 6.86- 6.84 (d, 1H, aromatic ring H), 0.90
(s, 18H, 2 × (CH3)3 of TBDMS), 0.14 (s, 6H, (CH3)2 of TBDMS), 0.13 (s, 6H, (CH3)2 of TBDMS).
f. 06. 7 Synthesis of compound 19
NaBH4 (32 mg, 0.85 mmol) was added in small portions to a solution of compound 18 (0.2501 g,
0.683 mmol) in dry THF (5 ml), refrigerated at 0°C thanks to an ice-bath. The reaction was stirred
for 2h and thirty at r.t., but we evaluated by monitoring the reaction with TLC that it did not go to
completeness, so we joined additional NaBH4 (7 mg, 0.17 mmol). Finally we treated the mixture
with dilute HCl and extracted the product with AcOEt. The organic layer was washed with water
until neutral pH, then dried with anydrous Na2SO4 and evaporated in vacuum.(0.2258 g, yield
90%).1H-NMR (400 MHz, CDCl3, 25 °C): δ = 6.87-6.82 (m, 3H, aromatic ring H), 4.57 (s, 2H,
CH2OH), 1.01 (s, 18H, 2 × (CH3)3 of TBDMS), 0.22 (s, 12H, 2 × (CH3)2 of TBDMS).
f. 06. 8 Synthesis of compound 13_TBDMS
We achieved the product 13_TBDMS drawing inspiration from a synthetic pathway reported in
literature26
. PBr3 (132 l, 1.22 mmol) was added dropwise to a solution of 19 (226 mg, 0.62 mmol)
in dry DCM (2 ml) at 0°C. After 30 min, the mixture was warmed to r.t. and magnetically stirred for
1 h. The crude was poured on ice and extracted three times with Et2O; the organic phase was dried
with Na2SO4 and evaporated. (0.218 g, yield 82%). 1H-NMR (400 MHz, CDCl3, 25 °C): δ = 6.89-
6.76 (m, 3H, aromatic ring H), 4.44 (s, 2H, CH2Br), 1.01 (s, 9 H, (CH3)3 of TBDMS), 1.00 (s, 9 H,
(CH3)3 of TBDMS), 0.22 (s, 12H, 2 × (CH3)2 of TBDMS).
88
f. 06. 9 Synthesis of compound 14_TBDMS
Also the product 14_TBDMS was obtained drawing inspiration from a method already reported in
literature27
. To a solution of ethylenediamine (1.69 ml, 25.3 mmol) in toluene (5 mL) was added
dropwise a solution of 13_TBDMS (0.218g, 0.506 mmol) dissolved in toluene (1 mL) over a period
of 25 min at r.t.. Then the reaction was heated up to reflux for 4 h followed by treatment of a water
solution of NaOH (90 mg in 400 L of H2O). After that the solvent and excess diamine were
evaporated in vacuum affording a residue which was taken up in DCM, washed with H2O and dried
on anhydrous Na2SO4.(0.1575 mg, yield 75%). 1H-NMR (400 MHz, CDCl3, 25 °C): δ = 6.78-6.76
(m, 3H, aromatic ring H), 3.68 (s, 2H, Ar-CH2NH), 2.83-2.80 (t, 2H, NHCH2), 2.69-2.63 (m, 2H,
CH2NH2), 0.98 (s, 9 H, (CH3)3 of TBDMS), 0.97 (s, 9 H, (CH3)3 of TBDMS), 0.18 (s, 12H, 2 ×
(CH3)2 of TBDMS). ESI-MS: m/z 411.5 [M+H]+.
f. 06. 10 Synthesis of the mono-catechol (c) co-surfactant: investigation of
reactivity of 14_TBDMS
We have designed the reaction reported in the Scheme 6 to investigate in detail why the coupling
between the compounds 14_TBDMS and 12 did not afford derivative 16.
.
OH
CH2OH
TBDMSO
TBDMSO
NHNH2
OH
CH2Br
OH
NHNH
OTBDMS
OTBDMS
X20 21
14-TBDMS
22
Scheme 6: Preparation of the derivative 22.
89
f. 06. 11 Synthesis of compound 21
The product 21 was achieved following the procedure described in the literature to achieve 4-(2-
Bromoethyl)phenol compound28
. The commercially acquirable 3-(4-hydroxyphenyl)propanol (250
mg, 1.64 mmol) was dissolved in 48 wt % HBr (1.3ml) affording a orange solution that was heated
up to 80°C and stirred for 16h. After that the reaction was warmed to r.t. and extracted with DCM.
The organic phase was dried on anhydrous Na2SO4, filtered off and finally evaporated. (0.3551g,
quantitative yield). 1H-NMR (400 MHz, CDCl3, 25 °C): δ = 7.09 (d, 2H, aromatic ring H), 6.80 (d,
2H, aromatic ring H), 3.41 (t, 2H; PEGCH2CH2OAr), 2.73 (t, 2 H, ArCH2), 2.19-2.10 (m, 2H,
CH2CH2CH2).
f. 06. 12 Synthesis of the mono-catechol (c) co-surfactant: acetonide
protecting group
OH
OH
COOH OH
OH
COOEt COOEtO
O
CH3
CH3
CH2BrO
O
CH3
CH3
CH2OHO
O
CH3
CH3
O
O
CH3
CH3
NHNH2
23 24 25 26
13_Acetonide 14_Acetonide
Scheme 7: Preparation of the derivative 14 with acetonide protecting group.
f. 06. 13 Synthesis of compound 24
We produced the compound 24 following the method reported in literature29
. Concentrated H2SO4
(80 l) was added dropwise to a solution of 3,4-dihydroxybenzoic acid (500 mg, 3.24 mmol) in
EtOH (7ml). Then the reaction was heated to reflux for 4h. Since by monitoring with TLC, we have
again observed the presence of starting material 23, we added additional 5 drops of concentrated
H2SO4 and we maintained the heating overnight. After evaporation of the solvent, the residue was
treated with water and the product extracted with diethyl ether. The organic phase was washed with
NaHCO3 water solution, water and salt, dried on Na2SO4 and evaporated. (405 mg, yield 69%). 1H-
NMR (400 MHz, CDCl3, 25 °C): δ = 7.26-7.23 (m, 2H, aromatic ring H), 6.63-6.61 (m, 1H,
aromatic ring H), 4.13-4.05 (m, 2H, OCH2CH3), 1.18-1.13 (m, 3H, OCH2CH3).
90
f. 06. 14 Synthesis of compound 25
We have followed the procedure described on a patent30
. A solution of 24 (200 mg, 1.10 mmol),
DMP (271 l, 2.20 mmol) and p-toluensolfonic acid as catalyst (8 mg) in dry toluene (15 ml) was
heated to reflux for 20h. Then toluene was removed and the residue was extracted with AcOEt,
washed respectively with a dilute water solution of NaHCO3, water and salt, dried on Na2SO4 and
evaporated. The crude was subjected to column chromatography (silica gel, hexane/EtOAc from
90/10 to 80/20) to give 25 as yellow solid (yield 49%).1H-NMR (400 MHz, CDCl3, 25 °C): δ =
7.63-7.60 (m, 1H, aromatic ring H), 7.41-7.39 (m, 1H, aromatic ring H), 6.77-6.72 (m, 1H, aromatic
ring H), 4.36-4.30 (m, 2H, COOCH2CH3), 1.70 (s, 6H, acetonide), 1.39-1.35 (m, 3H,
COOCH2CH3).
f. 06. 15 Synthesis of compound 26
According to the process detailed in the literature31
, a solution of compound 25 (140 mg, 0.63
mmol) in dry THF (3 ml) was added dropwise to a suspension of LiAlH4 (40 mg, 1.05 mmol) in
dry THF (3 ml), under N2, cooled at 0°C with an ice bath. Then the reaction was warmed to r.t. and
stirred overnight. The reaction was quenched with AcOEt, filtered on celite and purified by
chromatography (silica gel, hexane/EtOAc from 75/25 to 50/50), affording an oil (62.4 mg, yield
55%).1H-NMR (400 MHz, CDCl3, 25 °C): δ = 6.82-6.78 (m, 2H, aromatic ring H), 6.74-6.71 (m,
1H, aromatic H), 4.59 (s, 2H, CH2OH), 1.70 (s, 6H, acetonide).
f. 06. 16 Synthesis of compound 13_Acetonide
A solution of PBr3 (150 l, 1.58 mmol) in dry Et2O (2 ml) was added during 15 min to a solution
of 26 (0.26 g, 1.44 mmol) in dry Et2O (3 ml) at 0°C. Then the reaction was warmed to r.t. and
stirred for 30 min. The crude was poured on ice, quenched with water and stirred for 1h. Then the
product was extracted with DCM and the organic phase was washed with salt, dried with Na2SO4
and evaporated. (0.278 mg, yield 79%). 1H-NMR (400 MHz, CDCl3, 25 °C): δ = 6.85-6.80 (m, 2H,
aromatic ring H), 6.69-6.66 (m, 1H, aromatic H),4.49 (s, 2H, CH2Br), 1.70 (s, 6H, acetonide).
91
f. 06. 17 Synthesis of compound 14_Acetonide
A solution of 13-Acetonide (50 mg, 0.21 mmol) in dry THF (210 l) was added with a syringe
pump (8 l/min) to a solution of ethylenediamine (700 l, 10.5 mmol) in dry THF (800 l) at r.t.
After 1h, since the reaction was complete as monitored by TLC, the mixture was treated with
aqueous NaOH ( 10.4 mg, 0.26 mmol in 1 ml of water), and then extracted three times with DCM
(3 × 3 ml). The organic phase was dried with with Na2SO4, filtered off and evaporated in vacuum.
(33.6 mg, yield 72%). 1H-NMR (300 MHz, CDCl3, 25 °C): δ = 6.76-6.72 (m, 2H, aromatic ring H),
6.67 (d, 1H, aromatic ring H), 3.71 (s, 2H, Ar-CH2NH), 2.84-2.81 (t, 2H, NHCH2), 2.71-2.68 (m,
2H, CH2NH2), 1.68 (s, 6H, acetonide).
f. 06. 18 Synthesis of the mono-catechol (c) co-surfactant: benzyl ether
protecting group
CHOBnO
BnO
CH2OHBnO
BnO
CH2BrBnO
BnO
CH2ClBnO
BnO
CH2OMsBnO
BnO
27 28
13_BnO bromide
13_BnO chloride
13_BnO mesilate
Scheme 8: Preparation of different derivatives 13 with benzyl ether protecting group.
The derivative 13_BnO bromide was produced according with our previous work22
. The treatment
of compound 28 with SOCl2 or mesyl chloride allowed us to achieve respectively 13_BnO chloride
and 13_BnO mesilate.
92
f. 07. Preparation of PEG-ylated NPs
f. 07. 1 Preparation of PEG-ylated NPs from those obtained by hot-
injection
We have prepared 3 different batches of PEG-ylated NPs starting from 9.6 nm iron oxide NPs
prepared by hot-injection. A solution of PEG SS20_A or PEG SS33 (10.65 mg) or a mixture of both
of them (5.33 mg each) in CHCl3 (500 l) was added to 500 l of NP hexane solution (2.13 mg of
iron oxide NPs). The NPs was heated to 70 °C for 2 days, then precipitated with hexane, collected
by centrifugation and solved in deionized water (5 ml). The NPs were purified by dialysis for 2 days
giving a little precipitate that was left in the tube.
f. 07. 2 Preparation of PEG-ylated NPs from those obtained by co-
precipitation
3 ml of the water suspension of co-precipitated NPs (around 30 mg of Fe3O4) were treated with a
mixture of PEG SS20_A and PEG SS33 (107 and 119 mg respectively). Then they were stirred and
heated to 70 °C for 24h under N2.Finally NPs were solved in deionized water( final volume of 36.9
ml) and purified by dialysis for 1 day, frequently changing water.
93
g. Characterization of PEG-ylated NPs
g. 01. DLS of PEG-ylated NPs
Fig 15. DLS autocorrelogram (top) and intensity-weighted diameter distribution (bottom) of co-
precipitated NPs (M 448) coated with PEG SS20_A and PEG SS33. The mean diameter is 240 nm.
100
101
102
103
104
105
106
0.7
0.8
0.9
1
1.1
1.2
1.3x 10
8
/ s
C(
)
M448 10ug ml fil 0.2.dat
C()
22. Calculated Baseline.
23. Measured Baseline.
10-1
100
101
102
103
104
-1
0
1
2
3
4
5
6
7
8x 10
4WEIGHTED SOL.N for Intensity-Weighted Diameter Distribution (M448 10ug ml fil 0.2)
d (nm)
P
94
Fig 16. DLS autocorrelogram (top) and intensity-weighted diameter distribution (bottom) of
solvothermal NPs (M 415) coated with PEG SS20_A and PEG SS33. The mean diameter is 120 nm.
100
101
102
103
104
105
106
1.4
1.6
1.8
2
2.2
2.4
2.6x 10
8
/ s
C(
)
M415 filtratat.dat
C()
22. Calculated Baseline.
23. Measured Baseline.
10-1
100
101
102
103
104
-2
0
2
4
6
8
10
12
14
16
18x 10
4WEIGHTED SOL.N for Intensity-Weighted Diameter Distribution (M415 filtratat)
d (nm)
P
95
g. 02. CHN analysis data
Calculated Experimental
MeO - SS20A M415
% organic in the NPs
C 54,85% 40,41% 74%
H 9,09% 6,48% 71%
N 0% 0,09%
O 36,06%
Maleimm - SS33 M420
% organic in the NPs
C 55,08% 32,87% 60%
H 8,93% 5,21% 58%
N 0,77% 0,51% 66%
O 35,22%
Maleimm - SS33 & MeO - SS20A
M448 % organic in
the NPs
C 55,08% 50,62% 92%
H 8,93% 7,73% 87%
N 0,77% 0,33% 43%
O 35,22%
Fig 17. Results of the CHN analysis for solvothermal NPs (M415 and M420) and for co-
precipitated NPs (M 448) coated with PEG SS20_A and PEG SS33.
96
g. 03. Fluorescent assay to quantify maleimide groups
We reported in the Fig. 16 an example of calibration curve used to quantify maleimido groups by
fluorescence measurement.
Fig 18. Maleimide calibration curve
97
h. Characterization methods and instruments
1H-NMR spectra were registered with a Bruker Avance spectrometer in (
1H: 400 MHz or 300
MHz). 1H-NMR of PEG derivatives (around 10 mg of solid in 0.4 ml of deuterated solvent) were
collected with 32 scan and a recycle delay (D1 time parameter) of 10 s. HPLC purification was
performed by BiotageTM C18 reverse phase chromatography. Transmission electron microscopy
(TEM) images were taken with a Zeiss LIBRA 200FE microscope (Carl Zeiss AG, Oberkochen,
Germany). The TEM sample was achieved by evaporating in air a drop of diluted NP solution on a
carbon covered copper grid. FTIR spectra were registered with a Thermo Nicolet NEXUS 670
FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The sample for FTIR was
produced by grinding and pelleting dry NPs with KBr (NP:KBr 1:100 w/w). Hydrodynamic
diameter was measured employing a BI 90 Plus (Brookhaven Instruments Corporation, Holtsville,
NY, USA) at θ = 90°. Fluorescence measures were registered by a Synergy 2 Biotek Instrument.
98
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101
o Aknowledgments
Considero questo dottorato la conclusione di un periodo di ricerca iniziato 11 anni fa.
Da un punto di vista lavorativo un doveroso grazie va quindi in primis al Dott. Alessandro Ponti
che, in tutti questi anni trascorsi al CNR, è stato il mio responsabile scientifico.
Grazie anche alla Dott.ssa Alessandra Puglisi che, dopo un periodo trascorso lavorando assieme, ha
accettato da ricercatrice universitaria di svolgere il ruolo di mio tutor per questo dottorato.
Grazie agli “studiosi di scienza” con cui ho condiviso parte di questi anni in laboratorio: Alberto,
Alessandro, Anna, Claudio, Laura, Marcello, Marta, Primiano e Sandro.
E poi … grazie ai miei genitori … di tutto.
Grazie alle persone che in questi anni hanno fatto positivamente parte della mia vita.
Alle amiche storiche Gill e Marina.
A Silvia, che ha creduto ed investito nella nostra amicizia dimostrando che può nascere ovunque.
Alle amiche che ho conosciuto durante questi anni di lavoro: a Serena, la prima persona che vidi nel
sottoscala di via Golgi, lei che ancora spera che io sappia prendere la vita con più leggerezza e la
smetta di farle fare code in viaggio.
A Giusy, una forza della natura, che ogni volta mi ricorda che nella vita bisogna perseverare per
ottenere ciò che si vuole.
Ad Elena, (al grido di: “è viola,è viola!”), che mi ha voluto bene nonostante la sgridassi un giorno sì
e l’altro pure ( e che forse ora non ne ha davvero più bisogno).
A Cristina, carattere tosto sì , un po’come me, ma con un cuore grande e sincero.
A chi, se lo vorrà, entrerà davvero a far parte della mia vita …
Voi mi avete accompagnato in questi anni e spero continuerete a farlo: nei momenti belli ed in
quelli difficili che ci sono stati.
Voi mi avete fatto capire che l’amicizia, quella vera, resiste e che sono le persone, quelle speciali
come voi, che fanno la differenza e per cui vale la pena di impegnarsi, confrontandosi ogni giorno
per crescere assieme.
Vi voglio bene.
Sara
102
Ed ora, non potendo più storicamente ricoprire il ruolo di
faraone e neanche (per ora) quello di Papa, permettetemi
almeno di “fare la faraona”…
Example of final recipe:
Roast guinea fowl with chestnut, sage & lemon stuffing
Ingredients
1 small guinea fowl, about 1kg
8 rashers streaky bacon
50g soft butter
1 onion, unpeeled and thickly sliced
2 tbsp plain flour
350ml strong chicken stock
cranberry sauce, roast potatoes and vegetables, to serve
For the stuffing
1 onion, chopped
25g butter
1 tbsp chopped sage
50g walnut, finely chopped
50g breadcrumb
zest 2 lemons
¼ tsp ground mace
100g cooked chestnut, quartered
1 medium egg, beaten with a fork
103
Method
1. First make the stuffing. Soften the onion in the butter very gently, then stir in the sage and
cook for 2 mins more. Scrape into a bowl with the chopped walnuts, breadcrumbs, lemon
zest, mace, chestnuts and egg and mix together well. Season generously.
2. For the guinea fowl, wash and wipe out the inside cavity. Mix the butter with some
seasoning, then push and spread some under the skin over the breasts, and rub the rest over
the legs. Lay the bacon across the breasts, smoothing over, and season with some more
pepper. Push the stuffing into the cavity (any extra can be rolled into balls and baked in the
oven for the last 20 mins cooking time). You can cover and chill the guinea fowl now for up
to 24 hours.
3. To roast, bring the bird out of the fridge 30 mins before. Heat oven to 200C/180C fan/gas 6.
Sit the bird in a snug roasting tin with the sliced onion underneath. Roast for 15 mins, then
lower the oven to 180C/160C fan/gas 4 and roast for a further 35-45 mins for a 1kg bird (or
longer if bigger – use the timings for a roast chicken). Check the bird is done by piercing the
inside of the thigh with a knife and making sure the juices are clear, not bloody. Lift the
guinea fowl off the onions, onto a platter. Loosely cover with foil, top with a towel (to keep
it warm), and rest while you make the gravy.
4. Pour off the juices from the roasting tray into a jug or bowl, and allow to settle. Spoon a tbsp
of the fat on top back into the roasting tray, pop on the hob over a low heat (make sure your
roasting tray is suitable or transfer contents to a pan), and stir in the flour until it isn’t dusty
anymore. Gradually stir in the stock, plus any meat juices after you’ve discarded the rest of
the fat, and bubble gently until thickened. Season with salt, pepper, and pinches of sugar if it
needs it, then strain into a gravy jug and discard the onions. Serve with the guinea fowl,
spooning out the stuffing as you carve, plus cranberry sauce and plenty of vegetables.